brain injury - future medicine...future cience rou 919 stem cell therapies for traumatic brain...

Brain injury TOP ARTICLES SUPPLEMENT

CONTENTSEDITORIAL: Stem cell therapies for traumatic brain injury Regenerative Medicine Vol. 10 Issue 8

RESEARCH ARTICLE: Progenitor cell therapy for traumatic brain injury: effect of serum osmolarity on cell viability and cytokine production Regenerative Medicine Vol. 5 Issue 1

REVIEW: Progesterone treatment for brain injury: an update Future Neurology Vol. 5 Issue 1

EDITORIAL: Umbilical cord blood cell and granulocytecolony stimulating factor: combination therapy for traumatic brain injury Regenerative Medicine Vol. 9 Issue 4

http://www.futuremedicine.com/doi/pdf/10.2217/rme.15.62



http://www.futuremedicine.com/doi/pdf/10.2217/fnl.09.72



https://www.regmednet.com/

917Regen. Med. (2015) 10(8), 917–920 ISSN 1746-0751

part of

Editorial

10.2217/rme.15.62 © 2015 Future Medicine Ltd

Regen. Med.

Editorial 2015/08/3010

8

920

2015

Keywords: contusion • diffuse axonal injury • induced pluripotent stem cells • mononuclear cells • neural stem cells • neuronal precursors • neurotrophins • oligodendrocyte precursors • regeneration • stroke

The clinical problemTraumatic brain injury (TBI) is a common problem with unmet therapeutic needs. Although most cases of TBI are concussions that usually resolve over a few weeks, a great number of patients will suffer chronic dis-ability from TBI-associated encephalopa-thies. Such conditions include focal contu-sions related to low-impact falls and diffuse axonal injury (DAI) from ultrafast load-ing of axons due to rotational acceleration in the course of motor vehicle crashes and other scenarios. Focal contusions are impact injuries featured by intraparenchymal hem-orrhage with edema and ischemia in the inferior frontal and temporopolar regions leading to neuronal cell death and second-ary axonal degeneration. DAI is an impulse injury associated with dynamic loading of axons and represents the commonest neu-ropathology across TBI causes and degrees of severity. Clinical problems caused by traumatic contusions and DAI have no satisfactory treatments besides symptom-atic alleviation with physical/occupational/speech–language therapy and the empirical use of CNS-acting drugs. Clinical trials of small molecules have been unsuccessful [1]. Inspired by some earlier success in models of ischemic brain injury, stem cell transplan-tation has shown some preclinical efficacy, primarily in models of focal TBI. However, a number of limitations, including wide variance in transplanted cells and reported outcomes, make it difficult to draw general conclusions at this time.

Stem cells from bench to bedsideRecent discoveries of the ability of exogenous neural stem cells to become successfully incorporated into the neural parenchyma have refuted earlier notions on the mature nervous system as an environment unfavor-able to ongoing developmental events [2,3]. Such neural stem cells are derived from fetal neural tissue, embryonic stem cells or somatic cells induced to pluripotency with specific transcription factors. Scaling up produc-tion is challenging for fetal cells. Embryonic stem cells and induced pluripotent stem cell sources are theoretically inexhaustible and extremely pliable and have been induced to a large number of neuronal or glial fates includ-ing motor [4] and dopaminergic [5] neurons as well as oligodendrocytes [6]. Work with human neural stem cells in animal models was instrumental in the initiation of pioneer-ing clinical trials in motor neuron disease (NCT01348451, NCT01730716) and spinal cord injury (NCT01772810). Although it is too early to know the outcomes, these were landmark developments in regenerative neu-roscience and are already followed by early trials in other disorders. The therapeutic effects of neural stem cells in these condi-tions are presumably due to a combination of synaptic physiological actions and the syn-aptic release of neuroprotective molecules [7]. In an interesting turn of events, regenerative medicine based on stem cells may look more and more like regenerative medicine based on neurotrophins and trophic cytokines in the 1990s, perhaps with the added benefit that

Stem cell therapies for traumatic brain injury

Vassilis E KoliatsosAuthor for correspondence:

Department of Pathology, Division of

Neuropathology, The Johns Hopkins

University School of Medicine,

Baltimore, MD 21287, USA

and

Department of Neurology, The Johns

Hopkins University School of Medicine,


and

Department of Psychiatry & Behavioral

Sciences, The Johns Hopkins University

School of Medicine, Baltimore,

MD 21287, USA

[email protected]

Leyan XuDepartment of Pathology, Division of

Neuropathology, The Johns Hopkins

University School of Medicine,


Brian J CummingsDepartments of Physical & Medical

Rehabilitation, Neurological Surgery,

& Anatomy & Neurobiology, Sue &

Bill Gross Stem Cell Research Center,

University of California at Irvine, Irvine,

CA 92697, USA

“Inspired by some earlier success in models of ischemic brain injury, stem cell transplantation has shown some preclinical efficacy,

primarily in models of focal traumatic brain injury.”

For reprint orders, please contact: [email protected]

918 Regen. Med. (2015) 10(8) future science group

Editorial Koliatsos, Xu & Cummings

stem cells can produce these molecules indefinitely (Figure 1).

Preclinical modeling of TBIThe last 20+ years have seen considerable efforts in modeling TBI by cause or mechanism. These models are usually classified into focal and diffuse. Focal mod-els include the weight drop, controlled cortical impact injury and mid-line fluid percussion injury. Diffuse models are produced via inertial or impact accelera-tion. Modifications of fluid percussion injury have both focal and diffuse elements. These models have not been adequately tested in nude rats or SCID mice, in other words, subjects appropriate for stem cell transplantation, but this need has begun to be more recently addressed. Controlled cortical impact and impact acceleration models offer complementary opportunities for regenera-tive medicine: the former is a primary contusional injury with secondary axonal degeneration, whereas the latter is a model of DAI with secondary effects on neurons. Based on paradigms worked out in spinal cord injury, the previous models can be used to optimize neuronal- versus oligodendrocyte-based cell therapies: neuronal precursor transplants can be best optimized in models like controlled cortical impact, whereas oligodendrocyte precursor transplants can be best worked out in models of DAI. This contention does not imply that contusions are best treated with neuronal and DAI with oligoden-drocyte transplants. As in the case of spinal cord injury, both neurons and oligodendrocytes may need to be replaced and a mixed transplantation approach would probably work best in clinical TBI scenarios [8].

Stem cell transplantation as experimental therapy for TBIBecause of the complexity of TBI, we need to iden-tify specific targets for repair guided by pathological mechanisms. Such tasks may include replacing dead neurons or protecting injured axons and cell bod-ies and promoting axonal repair and regeneration (Figure 2). Neuronal degeneration or death is encoun-tered in both focal injury and DAI. Focal TBI (con-tusion) and DAI present different challenges in this regard: contusions may respond to targeted trans-plantation but cell death is acute; transplantation in DAI may need to be multifocal, but perikaryal degen-eration is slow and may or may not lead to cell death. Although axonal repair/remyelination as a therapeutic target is best established in spinal cord injury, demy-elination may also contribute to axonal degeneration in DAI [9]. Therefore, transplanting exogenous oligo-dendrocyte precursors in the case of DAI may assist in remyelination and prevent axonal degeneration and disconnection within brain circuits.

A growing number of studies with systemically administered stem cells may disclose novel mecha-nisms of neural injury and repair. Cells typically used in this approach are derived from bone marrow and include mesenchymal, multipotent adult and mono-nuclear stem cells. Bone marrow-derived stem cells are easy to access, require simple or no manipulation and have no attached immune rejection concerns if they are patient derived. Mononuclear cells have already entered several clinical trials: these cells are relatively small and thence not trapped in the lungs (first-pass effect) after intravenous administration, whereas less than 4% of intravenously injected mesenchymal cells reach arterial circulation [10]. Bone marrow-derived cells have shown efficacy in stroke models. Because of questionable penetration into brain, one of the pro-posed therapeutic mechanisms is the modulation of immune response [10]. Consistent with this view, there is growing evidence for the role of spleen as an organ that modulates neural injury [11]. The presumed tro-phic effects of systemically delivered bone marrow cells require further clarification. One study that has shown good blood–brain barrier penetration in controlled cortical impact injury also found an increase in lev-els of neurotrophins and VEGF in brain [12], whereas other studies have shown poor penetration [13]. This discrepancy may be due to first-pass effects or the particulars of experimental models. At any rate, it is unclear how trophic effects can be induced if these cells do not cross the blood–brain barrier [14].

Although parenchymal or intravenous administration of stem cells in models of TBI and stroke is common, intrathecal or intraventricular delivery is also being used

Figure 1. The progression from trophic factor therapies in the 1990s (left) to stem cell therapies in the 2000s (right) and now to the realization that part of stem cell efficacy may be mediated via release and transduction of trophic signals targeted and amplified via synaptic contacts (bottom). Adapted with permission from [21].

Evolution of trophic therapies for cell death in animal models in the 1990s

Stem cell efficacy in animal models mediated via trophic molecular mechanisms in addition to functional circuit formation

Stem cell transplants adopted to deal with some of the clinical pitfalls of trophic therapies

www.futuremedicine.com 919future science group

Stem cell therapies for traumatic brain injury Editorial

with some benefits. The potential of such strategies, especially the intraventricular route, for diffuse or multi-focal effects may be greater compared with that of paren-chymal strategies, especially if stem cells can be enticed to migrate to the lesion sites and differentiate into neu-ral cells [15]. However, the consistency and comparative advantages of such effects are far from established.

The outcomes of preclinical testing of stem cells in TBI models have been recently reviewed by one of us [16]. Positive effects were observed in most studies with a small mean effect size that was more pronounced with modified or ‘enhanced’ cells. Transplantation within the lesion (for focal TBI) had a larger effect size than intravenous or ventricular delivery. Unfortunately, many of these studies have methodological problems. In addition, there is as yet no common standard for the assessment of outcome measures. Furthermore, a syn-thesis of studies using different stem cell preparations is extremely difficult. Also, the majority of TBI studies using human stem cell do not assess cell survival, thus clouding our understanding of potential mechanisms of action. Although transplanted stem cells such as neu-ronal precursors are fully capable of forming mature synapses with host structures in the brain and spinal cord [17], the physiological status of these synapses and their specific role in restoring function has not been characterized. Functionality of regenerated synapses is important not only for the purpose of conveying appro-priate physiological signals but also for transsynaptic tro-phic support. The application of optogenetic strategies may prove critical in solving this problem [18].

Special considerations for focal TBI & DAIMost published stem cell experiments in TBI are on focal models: about half of them used some form of controlled cortical impact, and the rest are equally split between weight drop and fluid percussion. For reasons explained in the previous paragraph, the field is behind stroke and spinal cord injury. Although a variety of benefits have been reported, integration with the host has not been demonstrated. Also unresolved are the questions of optimal dosing and dose scaling to man; this is a tricky issue because, at least based on our expe-rience on spinal cord injury, dose escalation alters the dynamics of engraftment, migration and fate [19]. Even less can be said about stem cell therapies for models of DAI, although the field can borrow from spinal cord injury that invariably involves trauma in axonal tracts. In contrast to spinal cord injury, where axons course in restricted areas, DAI involves disparate axon tracts that would be difficult to transplant at the same time. Therefore, in the case of DAI, the choice of transplan-tation route (systemic, ventricular or parenchymal) and location of transplant (if we select parenchymal

delivery) are critical. A recent study has shown that, in sharp contrast to neuronal progenitors, human oligo-dendrocyte progenitors do not colonize and differen-tiate locally but rather migrate massively along white matter tracts and remain within the white matter, often ensheathing themselves around host axons [20].

ConclusionAfter multiple failures in clinical trials of single and combination agents, TBI is in dire need for effec-tive treatments. The nature of some of the key lesions invites the consideration of the toolkit of regenerative medicine, including stem cell transplants. Important advancements in preclinical stem cell therapeutics and the popularity of TBI models create unprecedented opportunities for discoveries that could push this stalled field forward. Although there is no lack of inter-esting data, a great disparity in models, cell prepara-tions, and reported outcomes detract from an enthu-siastic endorsement of stem-cell therapeutics for TBI at this time. Consortia to establish guidelines for TBI modeling and NIH initiatives supporting collaborative and replication platforms are urgently needed: it makes no sense to only fund original research that would make little difference in and of itself and would be difficult to replicate or integrate with other studies. In the course of experimenting with stem cell therapeutics, one of

Figure 2. Sketch of repair targets for stem-cell based therapies in traumatic brain injury. In upper panel, neuronal precursors support injured neurons (1) or, after they differentiate into nerve cells, they replace dead neurons (2). In lower panel, transplanted oligodendrocyte precursors support injured axons and prevent their degeneration (1) or facilitate the growth and maturation of axon sprouts (2). Immune signals related to systemically delivered bone marrow cells are not included.

1

1

2

2


Editorial Koliatsos, Xu & Cummings

the greatest promises is the discovery of physiological molecular signals that afford protection or promote recovery in the adult brain.

Financial & competing interests disclosureThis work was supported by a Maryland Technology Devel-

opment Corporation (TEDCO) grant 2015-MSCRF1-1718 to

VE Koliatsos. The authors have no other relevant affiliations

or financial involvement with any organization or entity with a

financial interest in or financial conflict with the subject mat-

ter or materials discussed in the manuscript apart from those

disclosed.

No writing assistance was utilized in the production of this

manuscript.

References1 Margulies S, Anderson G, Atif F et al. Combination

therapies for traumatic brain injury: retrospective considerations. J. Neurotrauma doi:10.1089/neu.2014.3855 (2015) (Epub ahead of print).

2 Cummings BJ, Uchida N, Tamaki SJ et al. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc. Natl Acad. Sci. USA 102, 14069–14074 (2005).

3 Yan J, Xu L, Welsh AM et al. Extensive neuronal differentiation of human neural stem cell grafts in adult rat spinal cord. PLoS Med. 4, e39 (2007).

4 Li XJ, Du ZW, Zarnowska ED et al. Specification of motoneurons from human embryonic stem cells. Nat. Biotechnol. 23, 215–221 (2005).

5 Perrier AL, Tabar V, Barberi T et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl Acad. Sci. USA 101, 12543–12548 (2004).

6 Hu BY, Du ZW, Zhang SC. Differentiation of human oligodendrocytes from pluripotent stem cells. Nat. Protoc. 4, 1614–1622 (2009).

7 Koliatsos VE, Xu LY, Yan J. Human stem cell grafts as therapies for motor neuron disease. Expert Opin. Biol. Ther. 8, 137–141 (2008).

8 Kwon BK, Sekhon LH, Fehlings MG. Emerging repair, regeneration, and translational research advances for spinal cord injury. Spine (Phila. Pa 1976) 35, S263–S270 (2010).

9 Flygt J, Djupsjo A, Lenne F, Marklund N. Myelin loss and oligodendrocyte pathology in white matter tracts following traumatic brain injury in the rat. Eur. J. Neurosci. 38, 2153–2165 (2013).

10 Cox CS Jr, Baumgartner JE, Harting MT et al. Autologous bone marrow mononuclear cell therapy for severe traumatic brain injury in children. Neurosurgery 68, 588–600 (2011).

11 Ajmo CT Jr, Vernon DO, Collier L et al. The spleen contributes to stroke-induced neurodegeneration. J. Neurosci. Res. 86, 2227–2234 (2008).

12 Mahmood A, Lu D, Chopp M. Intravenous administration of marrow stromal cells (MSCs) increases the expression

of growth factors in rat brain after traumatic brain injury. J. Neurotrauma 21, 33–39 (2004).

13 Harting MT, Jimenez F, Xue H et al. Intravenous mesenchymal stem cell therapy for traumatic brain injury. J. Neurosurg. 110, 1189–1197 (2009).

14 Koliatsos VE, Mocchetti I. Trophic factors as therapeutic agents for disease characterized by neuronal death. In: Cell Death and Diseases of the Nervous System. Koliatsos VE, Ratan RR (Eds). Humana Press, Totowa, NJ, USA (1997).

15 Lim JY, Jeong CH, Jun JA et al. Therapeutic effects of human umbilical cord blood-derived mesenchymal stem cells after intrathecal administration by lumbar puncture in a rat model of cerebral ischemia. Stem Cell Res. Ther. 2(5), 38 (2011).

16 Gold EM, Su D, Lopez-Velazquez L et al. Functional assessment of long-term deficits in rodent models of traumatic brain injury. Regen. Med. 8, 483–516 (2013).

17 Xu L, Ryugo DK, Pongstaporn T, Johe K, Koliatsos VE. Human neural stem cell grafts in the spinal cord of SOD1 transgenic rats: differentiation and structural integration into the segmental motor circuitry. J. Comp. Neurol. 514, 297–309 (2009).

18 Weick JP, Johnson MA, Skroch SP, Williams JC, Deisseroth K, Zhang SC. Functional control of transplantable human ESC-derived neurons via optogenetic targeting. Stem Cells 28, 2008–2016 (2010).

19 Piltti KM, Avakian SN, Funes GM et al. Transplantation dose alters the dynamics of human neural stem cell engraftment, proliferation and migration after spinal cord injury. Stem Cell Res. 15(2), 341–353 (2015).

20 Xu L, Ryu J, Hiel H et al. Transplantation of human oligodendrocyte progenitor cells in an animal model of diffuse traumatic axonal injury: survival and differentiation. Stem Cell Res. Ther. 6, 93 (2015).

21 Koliatsos VE, Xu L. Commentary on “the promise and the reality of stem-cell therapies for neurodegenerative diseases.” Cerebrum (2010). http://dana.org/Cerebrum/Default.aspx?id=39453

ReseaRch aRticle

ISSN 1746-0751 10.2217/RME.09.73 © 2010 Future Medicine Ltd Regen. Med. (2010) 5(1), 65–71

Progenitor cell therapy for traumatic brain injury: effect of serum osmolarity on cell viability and cytokine production

In the USA, 1.5 million patients suffer a trau-matic brain injury (TBI) each year, result-ing in 50,000 deaths with an additional 230,000 patients requiring hospitalization [1]. The overall prevalence of TBI is estimated to be 6.5 million people [2]. Of those patients affected, up to 48% are impaired by physical, cognitive and psychosocial deficits [3]. Beginning aggres-sive rehabilitation early in the patient’s hospital course has been shown to lead to improvement in functional status [4,5]; however, neurons show little ability to repair and no treatment modal-ity is currently available to reverse acute brain injury. A large body of work has failed to show significant efficacy from single-agent pharmaco-logic neuroprotective therapies. Therefore, the NIH recently convened a meeting to discuss the complex pathophysiology of neuronal injury and the failure of all trials based on current mono-therapies (controlled hypothermia, hyperosmotic infusion) to date. Recommendations included the development of in vitro models to research

multimodality treatments that could target sev-eral mechanisms of TBI’s complex pathophysio-logy [6,101]. Two therapeutic modalities currently under investigation for the treatment of TBI are the infusion of hypertonic saline (HTS) and _progenitor cell therapeutics.

Preliminary research has shown that HTS infusion after TBI could offer potential neuro-protection. One possible pathway could be via a decrease in intracranial pressure (ICP). Both preclinical research using a dog model [7] and prospective randomized trials [8] have shown decreased ICP after HTS infusion without sig-nificant effect on cerebral blood flow (CBF). Additional investigation completed by Khanna et al. demonstrated that HTS infusion in a pedi-atric population was associated with improved CBF in accordance with a decrease in ICP [9]. HTS resuscitation also increases mean arte-rial pressure, thereby attenuating a significant increase in poor neurological outcomes seen with hypotension within 6 h of TBI [10]. Furthermore,

Introduction: The potential translation of mesenchymal stem cell (MSC) therapy into a multimodal protocol for traumatic brain injury requires evaluation of viability and cytokine production in a hyperosmolar environment. Optimization of MSC therapy requires delivery to the target area without significant loss of cellular function or viability. No model evaluating the potential efficacy of MSC therapy at varying osmolarities currently exists. Methods: Rat MSCs were characterized with flow cytometric immunophenotyping. MSCs (passage 3) were placed in culture with multipotent adult progenitor cell media at varying osmolarities (250, 270, 290, 310, 330, 350 and 370 mOsm) potentially found with hypertonic saline infusion. After culture for 24 h, cellular viability was measured using flow cytometry (n = 6). Next, brain tissue supernatant was harvested from both normal rat brains and injured brains 6 h after cortical injury. Subsequently, MSCs were placed in culture with multipotent adult progenitor cell media ± 20% normal brain or injured brain supernatant (at the aforementioned osmolarities) and allowed to remain in culture for 24 h (n = 11). At this point, media supernatant cytokine levels were measured using a multiplex cytokine assay system. Results: MSCs showed no clinically significant difference in viability at 24 h. MSCs cultured with 20% injured brain supernatant showed an decrease in proinflammatory cytokine production (IL‑1a and IL‑1b) with increasing osmolarity. No difference in anti‑inflammatory cytokine production (IL‑4 and IL‑10) was observed. Conclusion: Progenitor cell therapy for traumatic brain injury may require survival and activity in a hyperosmolar environment. Culture of MSCs in such conditions shows no clinically significant effect on cell viability. In addition, MSC efficacy could potentially be enhanced via a decrease in proinflammatory cytokine production. Overall, a multimodal traumatic brain injury treatment protocol based upon MSC infusion and hypertonic saline therapy would not negatively affect progenitor cell efficacy and could be considered for multicenter clinical trials.

KEYWORDS: cytokines n inflammation n mesenchymal stem cells n osmolarity Peter A Walker, Fernando Jimenez & Charles S Cox Jr†

†Author for correspondence: Department of Pediatric Surgery, University of Texas Medical School at Houston, 6431 Fannin Street, MSB 5.234, Houston, TX 77030, USA Tel.: +1 713 500 7307; Fax: +1 713 500 7296 [email protected]

65


ReseaRch aRticle Walker, Jimenez & Cox Jr

Vialet et al. have shown HTS infusion to be more effective than mannitol as a hyperosmolar agent after TBI [11]. Despite promising initial results, the effect of HTS infusion upon ICP remains contro-versial as alternate prospective trials have failed to show improvement in ICP control when compared with crystalloid (lactated ringer’s) infusion [12,13]. HTS infusion decreased the number of compli-cations, the number of required interventions and length of intensive care unit stay after TBI in a pediatric population [14]; however, additional studies completed with an adult population failed to show a favorable effect on the required interven-tions [13]. Despite the promising results noted in the acute and subacute setting, some trials have failed to show sustained improved neuro logical outcomes 6 months after TBI [6,15]. As a result of the controversial results derived from the initial preclinical and clinical trials, the Joint Section on Neurotrauma and Critical Care did not make any recommendations for the use HTS infusion for TBI [16]; however, the potential neuro protection observed specifically in the pediatric population requires additional investigation.

A large amount of research has been com-pleted to investigate the potential role of progeni-tor (stem) cell therapeutics for the treatment of TBI. While initial preclinical research has shown a potential benefit from progenitor cell thera-peutics [17–20], the precise mechanism remains intensely controversial. Furthermore, the sec-ondary injury observed with TBI is associated with an induction of the systemic inflammatory response. Analysis of rat brain supernatant from a TBI model has shown significant increase in the proinflammatory cytokines IL-1a, IL-1b, IL-6 and TNF-a in both the direct injury and pen-umbral areas [21]. In addition, previous preclini-cal work has shown progenitor cells to migrate towards the site of injury and potentially modu-late cytokine production. Co-culture of mesen-chymal stem cells (MSCs) with purified immune cells has shown a decrease in proinflammatory cytokine production (TNF-a and IFN-g) with an increase in production of the anti-inflamma-tory cytokines IL-4 and IL-10 [22]. Therefore, MSCs could offer neuroprotection via modula-tion of locoregional proinflammatory cytokine production in accordance with the production of anti-inflammatory cytokines.

Previous research has shown HTS infusion and progenitor cell therapeutics to be ideal potential candidates for multimodal TBI treatment regi-mens. The potential translation of MSC therapy into such a multimodal protocol requires evalu-ation of cell viability and cytokine production in

a hyperosmolar environment. Previous research has shown osmotic stress to induce apoptotic cell death in multiple cell lines including mono-nuclear cells [23], cardiomyocytes [24] and fibro-blasts [25]. Furthermore, hyperosmotic stress has been shown to increase proinflammatory cyto-kine production from human peripheral blood mononuclear cells [26]. No model evaluating the potential efficacy of MSC therapy at vary-ing osmolarities currently exists. We designed a series of in vitro experiments to investigate MSC viability and cytokine production while cultured at the osmolarities observed with HTS infusion for the treatment of TBI.

Experimental designMesenchymal stem cells (passage 3) were placed in culture with multipotent adult progenitor cell (MAPC) media with the varying osmolarities (250, 270, 290, 310, 330, 350 and 370 mOsm) potentially found with HTS infusion. After 24 h in culture, cellular viability was measured using flow cytometry (n = 6 samples per media osmo-larity). Next, brain tissue supernatant was har-vested from both normal rat brains and injured brains 6 h after cortical injury. Subsequently, MSCs were placed in culture with MAPC media ± 20% normal brain or injured brain super natant (at the aforementioned osmolari-ties) and allowed to remain in culture for 24 h (n = 11 samples per media osmolarity). At this point, media supernatant cytokine levels were measured using a Bio-Plex cytokine assay system (Bio-Rad Laboratories, Hercules, CA, USA).

�n Ethical approvalAll protocols involving the use of animals were in compliance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Texas Institutional Animal Care and Use Committee (protocol HSC-AWC-07–055).

�n Data ana lysisUnless otherwise indicated, all values are rep-resented as mean ± standard error of the mean. Values were compared using ana lysis of variance (ANOVA) with a post-hoc Dunnett ana lysis using an osmolarity of 290 as the control. A p-value of ≤0.05 was used to denote statistical significance.

�n Isolation, characterization & labeling of rat MSCsMesenchymal stem cells were isolated from the bone marrow of Sprague-Dawley rats and expanded in MAPC media as previously

Regen. Med. (2010) 5(1)66 future science group

Stem cell viability & function in hyperosmolar conditions ReseaRch aRticle

described [27]. Flow cytometric immunopheno-typing was used to ensure that the MSCs were CD11b-, CD45-, CD29+, CD49e+, CD73+, CD90+, CD105+ and Stro-1+. Passage 3 cells were used for all experiments.

�n Measurement of media osmolarityThe osmolarity of MAPC media was mea-sured using the 5500 vapor pressure osmometer (Wescor Inc., Logan, UT, USA). Adjustments in media osmolarity were completed by addition of sterile distilled water or sodium chloride.

�n MSC viability Mesenchymal stem cells were removed from the culture plates and incubated with propridium iodide for 10 min. Using flow cytometry, the fraction of dead cells was calculated.

�n Controlled cortical impact injuryA controlled cortical impact (CCI) device (eCCI Model 6.3; VCU, Richmond, VA, USA) was used to administer a unilateral brain injury as described previously [28]. Male Sprague Dawley rats weighing 225–250 g were anesthetized with 4% isoflurane and a 1:1 mixture of N

2O/O

2 and

the head was mounted in a stereotactic frame. With the head held in a horizontal plane, a mid-line incision and subsequent 7- to 8-mm craniec-tomy was performed on the right cranial vault. The center of the craniectomy was placed at the midpoint between bregma and lambda, 3 mm lateral to the midline and overlying the tempo-parietal cortex. Animals received a single impact of 3.1-mm depth of deformation with an impact velocity of 5.8 m/s and a dwell time of 150 ms (moderate-to-severe injury) at an angle of 10° from the vertical plane using a 6-mm diameter impactor tip, making the impact orthogonal to the surface of the cortex. The impact was deliv-ered onto the parietal association cortex. Sham injuries were performed by anesthetizing the ani-mals, making the midline incision, and separat-ing the skin, connective tissue and aponeurosis from the cranium. The incision was then closed. The body temperature was maintained at 37°C by the use of a heating pad. Previously obtained serial arterial PaO

2 and PaCO

2 measurements

have shown that animals do not become hypoxic or hypercarbic during this procedure [21].

�n Brain homogenate supernatant fluid collectionRats were sacrificed 6 h after CCI or sham injury. Their brains were extracted and four regions, relative to the injury, were isolated: the site of

direct injury, penumbral region, ipsilateral frontal region and contralateral region, as we have previ-ously described [21]. The sections were weighed to ensure each section was 120 mg, gently minced with a pellet pestle, diluted in 1 ml (low-glucose Dulbecco’s modified Eagle medium with 10% fetal bovine serum; Gibco, Carlsbad, CA, USA), vortexed for 30 s and centrifuged for 6 min at 1000 g. The supernatant, containing intracerebral, interstitial fluid, was collected [21]. Of note, only the supernatant harvested from the direct injury region was used for stimulation of MSC cultures.

�n Cytokine ana lysisCytokines were detected in the MSC media super-natant using the Bio-Plex cytokine assay system (Bio-Rad Laboratories, Hercules, CA, USA). Concentrations of IL-1a, IL-1b, IL-4, IL-6, IL-10 and TNF-a were simultaneously evaluated using a commercially available multiplex bead-based immunoassay (Rat 9-Plex; Bio-Rad Laboratories). The assay was performed per the manufacturer’s instructions and the details have been previously published by our group and others [29,30]. High standard curves (low RP1 target value) for each soluble cytokine were used, ranging from 2 to 32,000 pg/ml. A minimum of 100 beads per cyto-kine region were evaluated and recorded. Values with a coefficient of variation beyond 10% were not included in the final data ana lysis. All samples were run in duplicate.


Figure 1. Mesenchymal stem cell viability measured after 24 h in multipotent adult progenitor cell media of varying osmolarities. Fractional viability values observed from 0.914 to 0.939 indicate that while there are statistically significant differences, adequate mesenchymal stem cell viability is observed under all culture conditions. *Statistical significance (one-way ANOVA with Dunnett’s post-hoc) compared with control osmolarity (290 mOsm) (p < 0.05).

1.00

0.98

0.96

0.94

0.92

0.90

0.88

0.86

0.84

0.82

0.80250 270 290 310 330 350 370 390

**

**

Fra

ctio

nal

via

bili

ty v

alu

es

Osmolarity (mOsm)


ResultsMesenchymal stem cells placed in culture with MAPC media at the various poten-tial osmolarities observed with HTS therapy (250–370 mOsm) showed a narrow range of viability (91.4–93.9%). Statistical ana lysis using an osmolarity of 290 to represent a physiologic control showed difference for osmolarities of

250, 330, 350 and 370. Figure 1 outlines MSC viability at the varying osmolarities.

Table 1 shows the proinflammatory cytokine production (IL-1a, IL-1b, IL-6 and TNF -a) measured in the media (with and without 20% normal/injured brain supernatants) of MSC cultures after 24 h. Table 1 shows a significant (p = 0.004) decrease in IL-1a production at

Table 1. Proinflammatory cytokine concentration (pg/ml) measured in the media (with and without 20% normal/injured brain supernatant) of mesenchymal stem cell cultures at varying osmolarities.

250 mOsm 270 mOsm 290 mOsm* 310 mOsm 330 mOsm 350 mOsm 370 mOsm

IL-1a

Media 18.5 ± 0.4 17.6 ± 1.0 18.0 ± 0.5 21.0 ± 1.6 19.3 ± 1.9 20.6 ± 1.6 19.1 ± 1.2

Normal brain supernatant

26.0 ± 0.4 30.1 ± 1.2 25.7 ± 0.8 33.2 ± 3.8 35.1 ± 3.2 38.1 ± 4.1 38.3 ± 6.2

Injured brain supernatant

399 ± 13.3 414 ± 10.6 448 ± 6.7 463 ± 31.3 452 ± 18.2 422 ± 10.3 375 ± 7.2*

IL-1b

Media 18.5 ± 0.4 17.6 ± 1.0 18.0 ± 0.5 21.0 ± 1.6 19.3 ± 1.9 20.6 ± 1.6 19.1 ± 1.2


33.3 ± 2.4 34.9 ± 2.6* 32.4 ± 1.3 38.9 ± 2.8 39.0 ± 2.2 39.8 ± 2.9 43.4 ± 4.1*


100 ± 3.7 92 ± 3.1 110 ± 6.5 108 ± 2.3 102 ± 3.7 100 ± 5.2 91 ± 4.8

IL-6

Media 6.8 ± 0.4 6.8 ± 0.5 6.5 ± 0.3 6.4 ± 0.2 5.8 ± 0.4 6.4 ± 0.2 6.4 ± 0.3


123 ± 33 131 ± 37 108 ± 28 110 ± 28 112 ± 29 123 ± 25 140 ± 31


178 ± 45 180 ± 49 185 ± 51 161 ± 42 160 ± 42 157 ± 46 162 ± 49

TNF-a

Media 7.0 ± 0.3 6.6 ± 0.2 7.0 ± 0.5 7.2 ± 0.5 6.8 ± 0.4 6.5 ± 0.2 6.9 ± 0.1


9.5 ± 0.3 9.8 ± 0.4 10.2 ± 0.3 9.9 ± 0.7 15.0 ± 2.4 16.2 ± 2.6 15.8 ± 2.9


10.1 ± 0.8 10.6 ± 0.6 10.9 ± 0.9 10.8 ± 0.4 9.9 ± 0.6 9.6 ± 0.7 9.2 ± 0.5

*Statistical significance (one-way ANOVA with Dunnett’s post-hoc) compared with control osmolarity (290 mOsm) (p < 0.05).

Table 2. Anti-inflammatory cytokine concentration (pg/ml) measured in the media (with and without 20% normal/injured brain supernatant) of mesenchymal stem cell cultures at varying osmolarities.

250 mOsm 270 mOsm 290 mOsm* 310 mOsm 330 mOsm 350 mOsm 370 mOsm

IL-4

Media 110 ± 3.6 103 ± 5.8 103 ± 2.2 107 ± 9.3 95 ± 7.8 119 ± 8.4 129 ± 9.1


213 ± 11.2 234 ± 10.4 207 ± 6.7 243 ± 16.6 222 ± 21.1 242 ± 17.6 252 ± 16.9


213 ± 34.7 220 ± 36.7 220 ± 39.3 204 ± 28.3 199 ± 23.5 184 ± 37.2 201 ± 35.6

IL-10

Media 2.6 ± 0.2 2.4 ± 0.2 2.5 ± 0.2 2.0 ± 0.0 2.6 ± 0.2 2.4 ± 0.2 2.4 ± 0.2


3.5 ± 0.2 3.3 ± 0.2 3.3 ± 0.3 3.1 ± 0.4 3.4 ± 0.2 3.3 ± 0.2 3.6 ± 0.4


3.7 ± 0.3 3.6 ± 0.3 4.4 ± 0.4 3.7 ± 0.3 4.0 ± 0.3 3.6 ± 0.4 3.5 ± 0.2

*Statistical significance (one-way ANOVA with Dunnett’s post-hoc) compared with control osmolarity (290 mOsm) (p < 0.05).



an osmolarity of 370 for MSCs cultured with injured brain supernatant (compared with the control value of 290). In addition, for MSCs cultured with injured brain supernatant there was a decrease in IL-1b production at serum osmolarities of 270 and 370 (p = 0.02) (Table 1). Further ana lysis failed to show a difference in the production of TNF -a or IL-6 when cultured in injured brain supernatant. In addition, no differ-ence was observed for MSCs cultured in media alone or with 20% normal brain supernatant.

Table 2 shows anti-inf lammatory cytokine production (IL-4 and IL-10) measured in the media (with and without 20% normal/injured brain supernatants) of MSC cultures after 24 h. No difference was observed for IL-4 or IL-10 at any osmolarity.

DiscussionMultimodal protocols for the treatment of TBI could require the intravenous infusion of MSCs with HTS therapy. Optimal cell efficacy requires adequate viability and function at elevated serum osmolarities. Our data show that MSC viability (91.4–93.9%) is largely unaffected by the range of serum osmolarities (250–370 mOsm) that could be observed in a traumatically injured patient. While a significant increase in MSC viability is observed with increasing osmolarity, difference in viability fails to reach clinical significance.

The potential neuroprotection observed with HTS therapy after TBI could be derived from a decrease in proinflammatory cytokine pro-duction. Recent investigation has shown an increase in plasma matrix metalloproteinases (MMPs) as well as IL-6 after TBI in both ani-mal models and injured patients [31,32]. MMPs are believed to contribute to further ischemic brain injury via breakdown of the blood–brain barrier. While initial work has shown controlled hypothermia to decrease the levels of MMP after TBI [32,33], little investigation into the effect of HTS has been completed. However, HTS therapy has been shown to mediate hepatic MMP production in a pancre-atitis model and could offer neuroprotection via a similar pathway after TBI [34].

The observed benefit from MSC therapy for TBI could be explained by modulation of the locoregional or systemic inflammatory milieu [17]. While still controversial, the implanted MSCs could be responsible for the produc-tion in anti-inflammatory cytokines that lead to enhanced neuroprotection. Our data show a decrease in production of the proinflamma-tory cytokines IL-1a and IL-1b with increasing

osmolarity. In addition, the production of the anti-inflammatory cytokines IL-4 and IL-10 remains unaffected with an increase in osmo-larity. These data show that increasing osmolar-ity does not affect MSC function in the form of anti-inflammatory cytokine production. In addition, increasing osmolarity may be of some benefit as evidenced by a decrease in the pro-inflammatory cytokine concentration, which could lead to enhanced neuroprotection.

ConclusionTraumatic brain injury is a major burden on the healthcare system worldwide. Current therapy in the acute setting is supportive with no pharmaco-logic treatment to attenuate neuronal cell death available today. The development of multimodal treatment protocols to attack TBI’s complex pathophysiology at multiple points could offer the most effective form of neuroprotection. The combination of HTS infusion and progenitor cell therapies could lead to improved CBF as well as modulation of the inflammatory response lead-ing to reduced neuronal death. Our data have shown that the intravenous infusion of MSCs into the hyperosmolar environment seen with HTS therapy has no clinically significant effect on cell viability. In addition, MSC efficacy could potentially be enhanced via a decrease in pro-inflammatory cytokine production. Overall, a multimodal TBI treatment protocol based upon MSC infusion and HTS therapy would not nega-tively affect progenitor cell efficacy and could be considered for multicenter clinical trials.

Financial & competing interests disclosureThe authors are supported by grants NIH T32 GM 08 79201, M01 RR 02558 R21, Texas Higher Education Coordinating Board, Children’s Memorial Hermann Hospital Foundation and The Brown Foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research The authors state that they have obtained appropriate insti tutional review board approval or have followed the princi ples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addi-tion, for investi gations involving human subjects, informed consent has been obtained from the participants involved.



Bibliography 1 Thurman DJ, Alverson C, Dunn KA et al.:

Traumatic brain injury in the United States: a public health perspective. J. Head Trauma Rehabil. 14(6), 602–615 (1999).

2 Consensus conference: Rehabilitation of persons with traumatic brain injury. NIH consensus development panel on rehabilitation of persons with traumatic brain injury. JAMA 282(10), 974–983 (1999).

3 Hawley CA, Ward AB, Magnay AR et al.: Outcomes following childhood head injury: a population study. J. Neurol. Neurosurg. Psychiatr. 75(5), 737–742 (2004).

4 Cowen TD, Meythaler JM, DeVivo MJ et al.: Influence of early variables in traumatic brain injury on functional independence measure scores and rehabilitation length of stay and charges. Arch. Phys. Med. Rehabil. 76(9), 797–803 (1995).

5 Gray DS, Burnham RS: Preliminary outcome ana lysis of a long-term rehabilitation program for severe acquired brain injury. Arch. Phys. Med. Rehabil. 81(11), 1447–1456 (2008).

6 Walker P, Harting MT, Baumgartner JE, Fletcher S, Strobel N, Cox CS Jr: Modern approaches to pediatric brain injury therapy. J. Trauma 67(Suppl. 2), S120–S127 (2009).

7 Pinto FC, Capone-Neto A, Prist R et al.: Volume replacement with lactated Ringer’s or 3% hypertonic saline solution during combined experimental hemorrhagic shock and traumatic brain injury. J. Trauma 60(4), 758–763; discussion 763–754 (2006).

8 Munar F, Ferrer AM, de Nadal M et al.: Cerebral hemodynamic effects of 7.2% hypertonic saline in patients with head injury and raised intracranial pressure. J. Neurotrauma 17(1), 41–51 (2000).

9 Khanna S, Davis D, Peterson B et al.: Use of hypertonic saline in the treatment of severe refractory posttraumatic intracranial hypertension in pediatric traumatic brain injury. Crit. Care Med. 28(4), 1144–1151 (2000).

10 Samant UB 4th, Mack CD, Koepsell T et al.: Time of hypotension and discharge outcome in children with severe traumatic brain injury. J. Neurotrauma 25(5), 495–502 (2008).

11 Vialet R, Albanese J, Thomachot L et al.: Isovolume hypertonic solutes (sodium chloride or mannitol) in the treatment of refractory posttraumatic intracranial hypertension: 2 ml/kg 7.5% saline is more effective than 2 ml/kg 20% mannitol. Crit. Care Med. 31(6), 1683–1687 (2003).

12 Qureshi AI, Suarez JI, Castro A et al.: Use of hypertonic saline/acetate infusion in treatment of cerebral edema in patients with head trauma: experience at a single center. J. Trauma 47(4), 659–665 (1999).

13 Shackford SR, Bourguignon PR, Wald SL et al.: Hypertonic saline resuscitation of patients with head injury: a prospective, randomized clinical trial. J. Trauma 44(1), 50–58 (1998).

14 Simma B, Burger R, Falk M et al.: A prospective, randomized, and controlled study of fluid management in children with severe head injury: lactated Ringer’s solution versus hypertonic saline. Crit. Care Med. 26(7), 1265–1270 (1998).

15 Cooper DJ, Myles PS, McDermott FT et al.: Prehospital hypertonic saline resuscitation of patients with hypotension and severe traumatic brain injury: a randomized controlled trial. JAMA 291(11), 1350–1357 (2004).

16 Brain Trauma Foundation; American Association of Neurological Surgeons; Congress of Neurological Surgeons; Joint Section on Neurotrauma and Critical Care, AANS/CNS; Bratton SL Chestnut RM, Ghajar J et al.: Guidelines for the management of severe traumatic brain injury. II. Hyperosmolar therapy. J. Neurotrauma 24, S14-S20 (2007).

17 Kim JM, Lee ST, Chu K et al.: Systemic transplantation of human adipose stem cells attenuated cerebral inflammation and degeneration in a hemorrhagic stroke model. Brain Res. 1183, 43–50 (2007).

18 Gao J, Prough DS, McAdoo DJ et al.: Transplantation of primed human fetal neural stem cells improves cognitive function in rats after traumatic brain injury. Exp. Neurol. 201(2), 281–292 (2006).

19 Mahmood A, Lu D, Wang L et al.: Treatment of traumatic brain injury in female rats with intravenous administration of bone marrow stromal cells. Neurosurgery 49(5), 1196–1203; discussion 1203–1194 (2001).

20 Lu D, Sanberg PR, Mahmood A et al.: Intravenous administration of human umbilical cord blood reduces neurological deficit in the rat after traumatic brain injury. Cell Transplant. 11(3), 275–281 (2002).

21 Harting MT, Jimenez F, Adams SD et al.: Acute, regional inflammatory response after traumatic brain injury: implications for cellular therapy. Surgery 144(5), 803–813 (2008).

22 Aggarwal S, Pittenger MF: Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105(4), 1815–1822 (2005).


Executive summary

Traumatic brain injury � Traumatic brain injury affects 1.5 million people per year in the USA. Of those affected, up to 48% have chronic physical, cognitive or

psychosocial deficits. No single modality pharmacologic treatment has shown efficacy.

Hypertonic saline therapy � Preliminary trials have shown potential neuroprotection via control of intracranial pressure with improvement of cerebral blood flow;

however, trials have failed to show cognitive improvement 6 months after injury.

Progenitor cell therapy � Initial in vivo models have shown potential neuroprotection based upon intravenous delivery; however, a review of the literature

shows no evidence of mesenchymal stem cell (MSC) viability at hyperosmolar conditions. Owing to NIH recommendations to develop multimodality therapeutic regimens for traumatic brain injury, a model to look at MSC viability and function is needed.

MSC viability � MSCs cultured at various osmolarities (250–370) showed no clinically significant difference in viability (91.4–93.9%).

Inflammatory cytokine production � MSCs showed a decrease in IL-1a and -1b production with increasing osmolarity. No difference in anti-inflammatory cytokine production

was observed. � Culture of MSCs at hyperosmolar conditions shows no clinically significant effect on cell viability. Additionally, MSC efficacy could

potentially be enhanced via a decrease in pro-inflammatory cytokine production.


23 Gastaldello K, Husson C, Dondeyne JP et al.: Cytotoxicity of mononuclear cells as induced by peritoneal dialysis fluids: insight into mechanisms that regulate osmotic stress-related apoptosis. Perit. Dial. Int. 28(6), 655–666 (2008).

24 Lee JW, Ko YE, Lee IH et al.: Osmotic stress induces loss of glutathione and increases the sensitivity to oxidative stress in H9c2 cardiac myocytes. Free Radic. Res. 43(3), 262–271 (2009).

25 Maeno E, Takahashi N, Okada Y: Dysfunction of regulatory volume increase is a key component of apoptosis. FEBS Lett. 580(27), 6513–6517 (2006).

26 Otto NM, Schindler R, Lun A et al.: Hyperosmotic stress enhances cytokine production and decreases phagocytosis in vitro. Crit. Care 12(4), R107 (2008).

27 Harting M, Jimenez F, Pati S et al.: Immunophenotype characterization of rat mesenchymal stromal cells. Cytotherapy 10(3), 243–253 (2008).

28 Lighthall JW: Controlled cortical impact: a new experimental brain injury model. J. Neurotrauma 5(1), 1–15 (1998).

29 Penolazzi L, Lambertini E, Tavanti E et al.: Evaluation of chemokine and cytokine profiles in osteoblast progenitors from umbilical cord blood stem cells by BIO-PLEX technology. Cell Biol. Int. 32(2), 320–325 (2008).

30 Adams SD, Radhakrishnan RS, Helmer KS et al.: Effects of anesthesia on lipopolysaccharide-induced changes in serum cytokines. J. Trauma 65(1), 170–174 (2008).

31 Hayashi T, Kaneko Y, Yu S et al.: Quantitative analyses of matrix metalloproteinase activity after traumatic brain injury in adult rats. Brain Res. 1280, 172–177 (2009).

32 Suehiro E, Fujisawa H, Akimura T et al.: Increased matrix metalloproteinase-9 in blood in association with activation of interleukin-6 after traumatic brain injury: influence of hypothermic therapy. J. Neurotrauma 21(12), 1706–1711 (2004).

33 Truettner JS, Alonso OF, Dalton Dietrich W: Influence of therapeutic hypothermia on matrix metalloproteinase activity after traumatic brain injury in rats. J. Cereb. Blood Flow Metab. 25(11), 1505–1516 (2005).

34 Rios EC, Moretti AI, de Souza HP et al.: Hypertonic saline reduces metalloproteinase expression in liver during pancreatitis. Clin. Exp. Pharmacol. Physiol. (2009) (Epub ahead of print).

�n Website101 NIH. Combination therapies for traumatic

brain injury workshop (2008). www.ninds.nih.gov/news_and_events/proceedings/Combination_Therapies_for_Traumatic_Brain_Injury_Workshop.htm

future science group 71www.futuremedicine.com

37ISSN 1479-6708

part of

Futu

re N

eu

rolo

gy

Future Neurol. (2010) 5(1), 37–4610.2217/FNL.09.72 © 2010 Future Medicine Ltd

BackgroundWhen our review of the role of progester-one (PROG) in brain injury was published in Future Neurology in 2006, there was no clinically available treatment for traumatic brain injury (TBI) [1]. This is still the case today. TBI is a major clinical concern in the USA and world-wide, and has been receiving increased public attention (and more funding) partly owing to the large number of concussive blast injuries suf-fered in the conflicts in Iraq and Afghanistan. US government sources indicate that, from 2003 to 2007, as many as 43,779 surviving combat casu-alties have been diagnosed with varying degrees of brain injury caused by explosive devices [101]. Taken together with more than 1.5 million annual cases of TBI occurring in the USA alone (including 300,000 annual sports-related injuries in children and young adults) [102], it is evident that brain injury continues to represent a substantial clinical and social problem.

Despite these grim data, there are grounds for optimism. Two independent Phase II clini-cal trials have now reported that PROG can be administered with a delay of up to 6 h or more after injury and still show beneficial results. The Progesterone for Traumatic Brain Injury, Experimental Clinical Treatment (ProTECT) trial was a randomized, double-blind, placebo-controlled trial with 100 patients suffering from moderate-to-severe brain injury (Glasgow Coma Scale [GSC] scores of 4–12) [2]. The patients treated with PROG were started on an intra-venous (iv.) drip of 0.71 mg/kg at 14ml/h for the first hour, reduced to 0.50 mg/kg at 10 ml/h for the next 11 h. Five additional 10-h infusions at 10 ml/h were administered for the remainder of the 3 days of treatment. No serious adverse

events were noted, and patients with severe inju-ries receiving 3 days of iv. PROG starting within 6–8 h after injury demonstrated a more than 50% reduction in mortality at 30 days compared with controls. The group with moderate injuries also showed significant ‘encouraging signs of improve-ment’ on their Disability Rating Scale (DRS) outcomes at 30 days compared with patients receiving placebo. The researchers concluded that PROG was beneficial for patients with both severe and moderate injuries, although the results were somewhat confounded in the severely injured by the fact that many in the group administered PROG survived who would not have otherwise survived without the treatment.

The ProTECT trial results were supported by another single-center trial of 159 severely brain- injured subjects (GCS score < 8) [3] that tracked patient outcomes for a longer period. The 82 subjects in the PROG group were treated for 5 days with intramuscular injections of PROG (1.0 mg/kg intramuscularly every 12 h for a total of 5 days) started within 8 h of injury and demon-strated significantly better survival as well as func-tional outcomes at both 3 and 6 months compared with the 77 patients given placebo. A dichoto-mized analysis demon strated that at the 3-month follow-up, 47% of the PROG-treated patients showed better functional outcomes on the GCS compared with 31% of the placebo group. At the 6-month follow-up the results were similar, with favorable outcomes in 58% of the PROG group compared with 42% of the placebo group. The PROG group had 18% mortality at 6 months, while the placebo group had 32% mortality at 6 months. It is important to emphasize that in both clinical trials PROG not only reduced mor-tality, but also significantly improved functional

Progesterone treatment for brain injury: an update

Milos Cekic & Donald G Stein†

†Author for correspondence: Clinic B, Suite 5100, 1365B Clifton Road NE, Emory University, Atlanta, GA 30322, USA n Tel.: +1 404 712 2540 n Fax: +1 404 727 2388 n [email protected]

Traumatic brain injury is a significant clinical problem for which there is still no effective treatment. Recent laboratory and clinical data demonstrate a potentially beneficial role for neurosteroids, such as progesterone and allopregnanolone, in the treatment of traumatic brain injury, ischemic stroke and some neurodegenerative disorders. Unlike single-target agents, progesterone affects many of the molecular and physiological processes in the cascade of secondary damage after a traumatic brain injury. This article updates a 2006 Future Neurology review of the research on progesterone and its metabolites in the treatment of traumatic brain injury, and presents new evidence that vitamin D deficiency can reduce progesterone neuroprotection, while combining progesterone with vitamin D produces better functional outcomes after TBI compared with eithertreatment alone.

Keywords

n functional recovery n neurosteroids n progesterone n traumatic brain injury

Revie

wFor reprint orders, please contact: [email protected]

Future Neurol. (2010) 5(1)38 future science group

Review Cekic & Stein

outcomes. Although these reports need to be con-firmed in larger multicenter trials, these two trials are the first pharmacological intervention for TBI to show a s ubstantial b enefit in human patients [4].

Based on these promising findings, the NIH is funding a national Phase III multicenter trial to test PROG in over 1000 moderately to severely brain-injured patients. This trial, termed ProTECT III, is designed to determine the effi-cacy of administering iv. PROG (initiated within 4 h of injury and administered for 72 h, followed by an additional 24-h taper) versus placebo for treating victims of moderate-to-severe acute TBI (GCS score of 12–4). The trial will track mortality, number of adverse and serious adverse events, DRS, and cognitive, neurological and functional outcomes.

Since the publication of the successful Phase II trials in 2007–2008 [2,3], there has been more interest in determining the extent to which PROG and some of its metabolites can enhance neuroprotection after different kinds of CNS injury. From just a few studies beginning in the 1990s, there are now over 100 published preclinical studies from 25 different laborato-ries, using four different species in 22 different injury models, demonstrating that PROG and its metabolites may be highly neuroprotective. Many of these studies have been published within the last few years.

Here we summarize recent findings illumi-nating some of the neuroprotective mechanisms of PROG and some of its metabolites. Further details can be found in extensive reviews by Brinton et al. [5], Schumacher et al. [6], Singh et al. [7], Stein and Hurn [8] and Stein [9]. The earlier literature on PROG focused mainly on the phenomenological markers of CNS repair so that, for example, if administered after a TBI, the hormone was shown to reduce cerebral edema, re-establish the compromised BBB, improve vascular tone, downregulate the expression of inflammatory factors, reduce excitotoxic damage and prevent post-traumatic seizures [5–10]. As these positive studies began to accumulate, attention turned to the question of how PROG was exert-ing its effects at the molecular level (whether this occurred through the c lassical PROG receptor or through other pathways).

Update on progesterone & recovery from brain injury: mechanisms & systems

OverviewOver two decades ago it was first noted that female rats show better recovery of cognitive function than their male counterparts after

bilateral damage to the medial frontal cortex (MFC). This effect was then found to vary with hormonal cycles and was due to endogenous lev-els of PROG, which in turn suggested the possi-bility of using exogenously administered PROG in order to improve recovery in both males and females [11]. Since then, the effectiveness of PROG treatment in functional recovery after TBI has been repeatedly demonstrated [5,6,9,12,13], with most of the work focusing on the mechanisms of action. We now believe that the neuroprotec-tive efficacy of PROG is a result of its actions on multiple genomic, proteomic and receptor sys-tems, rather than on a single mechanism. It has now also become more clearly established that moderate and severe TBI cause systemic injuries that alter metabolic and physiological functions throughout the body [14]; an effective treatment should therefore have s ystemic effects.

Molecular effects of progesterone A significant problem with damage to the CNS is disruption of blood flow to the local area of injury, resulting in loss of oxygen and glucose, energy failure and, ultimately, cell death. PROG has been demonstrated to protect neurons from ischemia and to decrease the size of the damaged area [8,15]. This resistance to injury is probably owing to several protective mechanisms, includ-ing maintenance of mitochondrial function, increased prosurvival signaling and reduced proapoptotic signaling [6,16–21].

PROG affects mitochondria in multiple ways. It helps restore them to normal morphology even after they have undergone severe vacuolation [22], inhibits the release of proapoptotic cyto-chrome c [23,24], and reduces the levels of pro-apoptotic B-cell lymphoma (Bcl)-2-associated X protein (Bax), Bcl-2-associated death-pro-moter (BAD) and activated caspase-3 [25–27]. PROG also upregulates the expression of antiapoptotic mitochondrial proteins such as Bcl-2. PROG may affect the expression of these mitochondrial proteins through activation of the ERK signaling pathway, which phosphory-lates the cAMP response element binding pro-tein (CREB), upregulates Bcl-2, and is known to provide improved resistance to ischemia [5]. PROG and its metabolites have also been demon strated to modulate MAPK and PI3K sig-naling in the hippocampus, hypothalamus and cerebellum of ovariectomized rats in vivo [28,29]. Furthermore, PROG has been demonstrated to reverse alterations in mitochondrial respi-ration [30] and to normalize the expression of Na+,K+-ATPase in experimental autoimmune


Progesterone treatment for brain injury: an update Review

encephalomyelitis and models of nerve and spi-nal cord crush injury [31–33]. Since both respira-tion and Na+,K+-ATPase are important compo-nents in the cascade that leads to energy failure and loss of ionic gradients, we believe that this regulation of cellular metabolism is a key step in the prevention of cell death and the attenuation of secondary injury.

PROG has also been demonstrated to reduce inflammation, a significant mechanism of sec-ondary injury, and is known to reduce microglial activation, as well as production of proinflam-matory cytokines such as TNF-a and IL-1 [9,34]. This reduced inflammatory response is not only beneficial in the injury penumbra but has sig-nificant systemic effects, since TBI-associated systemic inflammation can often lead to multi-organ failure and infection [13] (mechanisms that are, in addition to cerebral edema, a frequent proximal cause of death after brain injury). Similar findings have been reported by Pan and colleagues, who found that following TBI in rats, expression of NF-kb, p65 and TNF-a were markedly decreased if the animals were adminis-tered PROG injections soon after the injury [35]. Measurement of brain edema, lesion volume and behavioral outcomes also revealed that PROG was effective in reducing the severity of the dam-age. Furthermore, in a series of studies, Chen et al. demonstrated that treatment with PROG reduces the expression of inflammatory cyto-kines, not just in the damaged brain, but also systemically in non-neuronal tissue such as the gut, spleen and intestine [17,36–38]. These findings support the systemic nature of brain injury and the fact that PROG acts at multiple receptor sites to control inflammation and improve functional outcomes (see also [39,40]).

Furthermore, there is evidence that PROG treatment after TBI reduces lipid peroxidation. The mechanisms of this action are not com-pletely understood [6], but it is thought to occur in the upregulation of antioxidant enzymes such as superoxide dismutase [41]. Reducing the dam-age caused by reactive oxygen species and nitro-gen species is known to improve cell survival in the penumbra of the injury by maintaining membrane integrity, and also helps to maintain the BBB by limiting oxidative damage to the cap-illary endothelium. PROG also helps maintain BBB function by upregulating P-glycoprotein, an efflux pump transporter and marker of BBB health that serves to eliminate xenobiotic and toxic substances, which consist of inflammatory cytokines and reactive oxygen species-producing compounds in the case of traumatic injury [42].

These molecular mechanisms – decreased inflammation and lipid peroxidation, mainte-nance of BBB integrity and improved ionic sta-bility – all help to reduce cerebral edema after TBI [6]. Recent findings also indicate that PROG regulates the expression of aquaporin-4, a water channel expressed in astrocyte endfeet that is thought to be important for the development of edema [43]. Since brain swelling is one of the final neurological causes of mortality after TBI, this is an important concern for the clinical m anagement of patients with brain injury.

Summary of progesterone effects on receptorsAlthough many of the molecular effects of PROG in neuroprotection have been described, it is still not known whether the classical intra-nuclear receptors are responsible for all of the hormone’s effects. In this review and others, perhaps contrary to the opinions of colleagues convinced that PROG can act only at a single receptor site, we have stressed the hypothesis that PROG has multiple beneficial effects that cannot be attributed solely to the PROG intra-nuclear receptor. For example, VanLandingham et al. have demonstrated that the enantiomer of PROG can substantially reduce cerebral edema following TBI without being able to bind to PROG receptor [44]. PROG and its metabolites, such as allopreganolone, can reduce excitotox-icity at the NMDA receptor [45] and possibly decrease the incidence and severity of post-trau-matic epilepsy by acting to potentiate g-amino-butyric acid type A (GABA

A) receptors to release

more inhibitory neuro transmitters, thus acting, in some respects, similar to barbiturates and other anesthetics (e.g., see [39]). Studies in PROG receptor-knockout mice have demonstrated that the hormone can still exert anxiolytic and anes-thetic effects, even in the absence of the PROG receptor. This probably occurs through the hor-mone’s metabolism to allopregnanolone and its actions on the GABA

A and s-1 receptors [46–48].

Covey and associates have published excellent and detailed reviews describing how neuro steroids interact with the GABA

A receptor sites [49]

(see [50] for more detail). With regard to mediat-ing inflammatory reactions, PROG utilizes the glucocorticoid and PROG receptors to inhibit the production of nitrite and cytokines, such as IL-12, whereas synthetic progestins do not have this effect [51]. After brain injury, it also appears that PROG can modulate the activation of Toll-like receptors, which in turn affect the expres-sion of inflammatory signaling factors, such as



NF-kb and other pro inflammatory cytokines [36]. In neuro protection, intracellular calcium regula-tion and homeostasis are important for cell sur-vival after injury. Hwang et al. found that PROG influences the activity of inositol 1,4,5-trisphos-phate receptors, which in turn regulate the levels of intracellular calcium in cultures of primary hippo campal neurons [52]. Some researchers argue that PROG can have both inhibitory and excit-atory actions depending on whether the hormone is acting in the peripheral nervous system or CNS. For instance, Viero and Dayanithi demonstrated that PROG could produce substantial calcium influx by activating GABA

A and oxytocin recep-

tors in the hypothalamus and supraoptic nucleus in neonatal rodents, but they observed the oppo-site effects in embryonic dorsal root ganglion, where GABA inhibited calcium influx [53]. The very rapid effects of PROG (such as its effects on inflammation and edema) are likely to be non-genomic and triggered at the cell surface. Recent experiments have identified a number of mem-brane PROG receptors, such as 25-Dx, or mem-brane PROG receptor-a, which when activated can stimulate the formation of new synapses and dendrites – important components of repair after CNS damage [54]. Guennoun et al. demonstrated that 25-Dx expression is substantially increased in neurons and astrocytes after both spinal cord and brain injuries, whereas the classical intranuclear receptor was actually downregulated [55]. It is also interesting to note that the 25-Dx receptor is also very abundant in the hypothalamus, where growth hormone expression plays a role in CNS repair in response to cerebral injury.

From even this very cursory review of PROG receptor mechanisms, it is apparent that neuro-steroid receptor mechanisms and their role in CNS repair are complex. It is well beyond the scope of this update to provide full details, but some excellent reviews are available [5,21,56–58].

Progesterone in aging & brain injuryAlthough TBI affects all age groups, it is an especially significant health problem for those aged over 70 years, where both the incidence of hospitalizations and mortality rates due to TBI are highest [59]. While deaths resulting from TBI have been reduced in most demographics with improvements in safety, they have increased sig-nificantly in the older population. The elderly are subject to physiological and metabolic altera-tions that can affect recovery after major trauma, and therefore need to be considered specifically with regard to both treatment modalities and characteristic underlying physiology.

Something new to consider: traumatic brain injury, progesterone, aging & vitamin D deficiencyIn a recent study, our laboratory tested the effec-tiveness of PROG in attenuating the acute inflam-matory response after TBI in aged rats [42]. We observed significantly reduced levels of inflamma-tory markers, such as TNF-a, IL-6, NF-kb-p65 and COX-2, at 24, 48 and 72 h after injury. In addition, PROG reduced cell death, as measured by caspase-3 levels, and improved BBB integrity, as measured by P-glycoprotein levels. These molecular data were correlated with reduced cerebral edema and improved functional activ-ity, all of which was consistent with the beneficial effects of PROG after TBI recorded repeatedly in younger animals [17,25,26,30,35,36,37]. The optimal doses and duration of treatment were also similar to those observed in younger animals, suggesting that PROG and its meta bolites may be effective as a treatment for TBI across the life cycle.

In addition to advanced age itself, other fac-tors that may affect the severity of TBI in the elderly include systemic issues, such as cardio-vascular disease and atherosclerosis, hyper-tension, diabetes, kidney disease, cancer and hyperparathyroidism [60]. All these conditions can individually affect the response to injury, and each has also been recently associated with insufficient serum levels of vitamin D [61,62], suggesting that vitamin D deficiency may pose a risk factor for TBI severity in the elderly. This is important since vitamin D deficiency has been noted in all segments of the population, but especially in the elderly [61] and hospitalized individuals [63,64].

In another recent study, we examined the effects of vitamin D deficiency on acute inflammation in aged rats after TBI [65]. Our results demonstrated that, in both injured and uninjured aged rats, vitamin D deficiency can significantly elevate the levels of inflammatory markers (TNF-a, IL-1b, IL-6 and NF-kb-p65), increase cell death and affect short-term behavior. This acute effect is important in translating these results to the human population since acute inflammation has been strongly linked to survival in human trauma patients [66]. Surprisingly, vitamin D deficiency also attenuated the beneficial effects of PROG administration after TBI, although it was possible to overcome this through coadministration with 1,25-dihydroxyvitamin D

3 (VDH), the active

form of vitamin D (Figure 1). Considering the prevalence of vitamin D deficiency in the elderly human population, these results could have sig-nificant consequences for clinical practice. They



also suggest that treatments with multiple drugs that affect different mechanisms of CNS injury and repair may be effective in the management of TBI and other CNS disorders.

Progesterone & combination therapiesThe past two decades have seen the failure of many Phase II and III clinical trials for moderate and severe TBI, despite the fact that over 130 dif-ferent compounds had initially shown promise in preclinical models of injury [67]. One of the major reasons suggested for this disappointing state of affairs is the fact that the complex and varied mechanisms observed in TBI cannot be adequately addressed by single-drug therapies targeted to a specific mechanism or receptor site. In contrast to the clear outcomes and well-circumscribed injuries of experimental animal models of injury, clinicians are well aware that human TBI pathophysiology is highly variable and heterogeneous, and affects many organs and tissue systems outside the brain itself. There is growing recognition that pharmacotherapies tar-geting more than one mechanism, or the same mechanisms differently, or at different time-points, may be more effective than the commonly applied ‘monotherapies’ [67]. This idea is already prevalent in the treatment of diseases such as HIV/AIDS and tuberculosis, where multithera-peutic approaches that target different parts of the disease process, act at different sites, and

synergistically increase activity have become the standard. A similar approach has been suggested for TBI owing to its complex manifestation in human patients [67]. A number of treatments other than PROG have been suggested as the ‘main’ component of a potential combination therapy, including citicoline, erythropoietin, hypo thermia, cyclo sporin A, statins and hyper-tonic saline [67], all of which have shown some promise in preclinical experiments. A literature review and our previous data with vitamin D deficiency suggest that VDH may also be a good candidate for combination therapy [68]. VDH is a neuroactive steroid known to be neuropro-tective, with functional attributes similar to PROG, although it also has a number of diver-gent actions. It is also cheap, easy to administer and readily available. Based on our laboratory data, we think that a combination of PROG and VDH could lead to improved neuronal and cellu-lar repair and recovery after injury, perhaps with less dosing and duration of treatment than with either agent administered alone.

A recent paper from our laboratory supports this approach [69]. We found that in cultured cortical neurons challenged with glutamate, both VDH and PROG individually demonstrated neuro-protection, but combined treatment was more effective than treatment with either compound alone. Significantly, the most effective combina-tion dosages were different from the individual

SHAM VH PROG SHAM VH PROG D + PROG D

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Rat

io o

f p

ost

inju

ry:p

rein

jury

loco

mo

tio

n

‡*

‡*

‡*

‡*

Figure 1. Effects of vitamin D deficiency on short-term behavior after traumatic brain injury. Locomotor behavior associated with acute inflammation in aged rats 72 h after TBI. The dark bars represent vitamin D-sufficient animals and the light bars represent vitamin D-deficient animals. It should be noted that only a combination of PROG (16 mg/kg) and VDH (5 mg/kg; D+PROG) returns behavior to sham-injured levels in deficient animals, whereas PROG alone is sufficient in nutritionally normal animals. This suggests that vitamin D deficiency exacerbates the injury and interferes with PROG treatment, but also that this effect can be reversed with acute VDH administration. *p < 0.05 versus sufficient VH; ‡p < 0.05 versus deficient VH.PROG: Progesterone; TBI: Traumatic brain injury; VDH: 1,25-dihydroxyvitamin D

3; VH: Vehicle-treated group.

Adapted with permission from [65].



‘best’ doses, suggesting synergy between the two drugs that would not be predicted based on t reatment with either agent alone (Figure 2).

One reason for treating injury pathways with multiple compounds is the possibility that a sin-gle repair mechanism may be modulated through different signaling mechanisms. Another reason is that a single high-dose drug treatment could saturate receptors and result in loss of functional benefit, which can happen in the case of PROG, for example [70]. This problem might be over- This problem might be over-come by activating a different pathway leading to neuroprotection [71]. As an example, PROG and VDH could each increase the activity of g-glu-tamyl transpeptidase by different mechanisms (PROG receptor–PROG activity and vitamin D receptor–VDH activity), resulting in higher over-all antioxidant capacity. The possibility of ampli-fying a neuroprotective effect by c ombinatorial therapy is worth further exploration.

In summary, there is now substantial preclini-cal and clinical evidence that PROG can have salutary effects on morphological and functional recovery after TBI. Over the last 5–6 years and even since the first review of PROG in Future Neurology, much has been discovered about its specific mechanisms of repair. A comprehensive discussion of the many receptor mechanisms involved in neurosteroid and vitamin D actions is beyond the scope of this update, but there are

a number of recent articles on this subject [71–76]. It is unfortunate that, thus far, most clinical trials for TBI treatments have failed, leading to con-siderable pessimism that a successful treatment will ever be found. Nonetheless, the fact that the NIH is supporting a Phase III multicenter trial using PROG for moderate-to-severe TBI is encouraging [103]. Unlike all other agents recently tested, PROG is a naturally occurring hormone with a high safety profile as demonstrated in two Phase II trials, both of which have shown substantial benefit in reducing mortality and improving functional outcomes after TBI [2,3].

Future perspectiveAs the Phase III trial goes forward, investiga-tors and clinicians are asking: what’s next? One of the key clinical areas now under investigation in our laboratory is ischemic stroke. There is a small amount of literature demonstrating that in animal models of ischemic injury, PROG and its metabolite allopregnanolone can reduce the size of the ischemic infarct and produce func-tional benefits [6]. Less is known about PROG in the treatment of military blast injuries to the head and in pediatric traumatic or hypoxic brain injury, although these are also important poten-tial applications. There is also growing interest in determining whether neurosteroids could be applied to more chronic neural disorders, such as

CTRL VH P20 P+D1 P+D5 P+D10 P+D20 P+D40 P+D80 P+D100

0

20

40

60

80

100

120

Cel

l su

rviv

al (

% C

TR

L)

§

*

‡ ‡

‡ ‡

‡

‡

‡

‡

Figure 2. Effects of combination progesterone and 1,25-dihydroxyvitamin D treatment on cell survival in vitro. The effects of progesterone and VDH combination treatment on glutamate-induced MTT reduction in rat primary cortical neurons. Cells were pretreated with different combinations of progesterone (20 µM) and VDH (doses shown as ‘D’ nM) for 24 h and subsequently exposed to glutamate (0.5 µM) for 24 h. Note the significant synergistic effect between P and D20, which is not observed with all combination dosages. *p < 0.001 versus VH; ‡p < 0.001 versus CTRL; §p < 0.01 vesus P20 alone. CTRL: Control; P: Progesterone; VDH: 1,25-dihydroxyvitamin D; VH: Vehicle. Adapted with permission from [69].



Executive summary

BackgroundnRecent data demonstrates that neurosteroids, specifically progesterone (PROG) and some of its metabolites, are neuroprotective in

experimental models of traumatic brain injury (TBI). nTwo recent Phase II clinical trials of PROG as a treatment for TBI have also shown promise. A multicenter Phase III trial is now underway

to determine clinical effectiveness.

Mechanisms & systemsnPROG has been demonstrated to protect neurons from ischemic injury and to decrease the size of a lesion or ischemic infarct. These

actions are a result of the hormone’s effects on maintenance of mitochondrial functions, increased prosurvival signaling, reduced inflammatory reactions, apoptosis and reactive oxygen species, stimulation of myelin synthesis and restoration of the BBB. These effects are systemic and not limited to the CNS injury itself.

ReceptorsnPROG acts at a number of different sites to enhance neuroprotection.nPROG and its metabolites can upregulate gene activity through their direct actions on the PROG intranuclear receptor, but they can also

act at the NMDA receptor to reduce excitotoxicity and at the GABAA and s-receptors to release more inhibitory neurotransmitters and

thus block post-traumatic seizure activity. nPROG modulates Toll-like receptors, and this action affects the expression of inflammatory factors and cytokines such as NF-kb and

IL-1, among others. nThe hormone also acts on membrane receptors, such as 25-Dx, which alters calcium toxicity.

Progesterone, aging & vitamin D nVitamin D deficiency, especially in older subjects, leads to increased expression of inflammatory factors and reduces the benefits of

PROG treatment for TBI. Combining vitamin D with PROG leads to better functional outcomes than either treatment alone.nBoth PROG and vitamin D exert their effects through a variety of metabolic pathways and receptor mechanisms activated in the

secondary injury cascade after TBI. Both can promote neural survival, repair and better functional outcomes.

Progesterone & combination therapies nMany clinical trials have failed because they do not address the complexity of TBI as a systemic disease and the varied mechanisms involved

in tissue damage and repair. There is growing recognition that combination therapies, such as vitamin D and PROG, can have more beneficial effects in combination than when administered alone. This is a relatively new area of research that requires more attention.

Future perspectivenThe effects of neurosteroids on TBI may also prove beneficial in several other applications. In all cases, much work remains to be done:

– Ischemic stroke: a few recent studies in animal models suggest that PROG and allopregnanolone can reduce infarct size and produce functional benefits; – Pediatric traumatic or hypoxic brain injury: some animal models are now being studied.

nPROG’s effects on remyelination and inflammation may make it a therapeutic candidate for neurodegenerative and other chronic CNS disorders, such as amyotrophic lateral sclerosis, multiple sclerosis and Parkinson’s disease. This is a very new area where work has only just begun.

amyotrophic lateral sclerosis, multiple sclerosis or Parkinson’s disease. This is certainly a topic for fur-ther review and discussion, but far too p reliminary at this stage to consider for clinical trial.

A key point to keep in mind is that this hor-mone has characteristics that are protective of the fetus during gestation and of the nervous system when administered exogenously throughout the spectrum of development, including old age. It can be argued that PROG works as a neuro-protective agent because, to a great extent, many of the processes involved in brain repair are simi-lar to growth and organizational processes occur-ring in early life. This notion is admittedly debat-able, but it is a hypothesis that can be subjected to experimental testing and verification. If only because not much else in the field of brain injury has been shown to work as well, the hormone and its related metabolites deserve continued e xperimental attention and clinical evaluation.

Financial & competing interests disclosurePortions of the research described in this manuscript were supported by NIH grants 5R01HD061971 and 5R01NS048451. Donald Stein is entitled to royalties from products of BHR Pharma related to the research described in this presentation, and may receive research funding from BHR, which is devel-oping products related to this research. In addition, the author serves as consultant to BHR and receives compensation for these services. The terms of this arrangement have been reviewed and approved by Emory University in accordance with its conf lict of interest policies. The authors have no other relevant af f iliations or f inancial involvement with any organization or entity with a financial interest in or f inancial conf lict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the p roduction of this manuscript.



BibliographyPapers of special note have been highlighted as:n of interestnn of considerable interest

1. Stein DG: Progesterone in the experimental treatment of central and peripheral nervous system injuries. Future Neurol. 1(4), 429–438 (2006).

2. Wright DW, Kellermann AL, Hertzberg VS et al.: ProTECT: a randomized clinical trial of progesterone for acute traumatic brain injury. Ann. Emerg. Med. 49, 391–402 (2007).

nn Along with [3], presents results of Phase II trials using progesterone to treat moderate-to-severe traumatic brain injury (TBI).

3. Xiao G, Wei J, Yan W, Wang W, Lu Z: Improved outcomes from the administration of progesterone for patients with acute severe traumatic brain injury: a randomized controlled trial. Crit. Care 12, R61 (2008).

nn Along with [2], presents results of Phase II trials using progesterone to treat moderate-to-severe TBI.

4. Doppenberg EM, Choi SC, Bullock R: Clinical trials in traumatic brain injury: lessons for the future. J. Neurosurg. Anesthesiol. 16, 87–94 (2004).

5. Brinton RD, Thompson RF, Foy MR et al.: Progesterone receptors: form and function in brain. Front. Neuroendocrinol. 29, 313–339 (2008).

nn Excellent overview of progesterone receptor mechanisms in the CNS.

6. Schumacher M, Guennoun R, Stein DG, De Nicola AF: Progesterone: therapeutic opportunities for neuroprotection and myelin repair. Pharmacol. Ther. 116, 77–107 (2007).

n Provides a detailed review of the potential uses of progesterone in the treatment of a spectrum of CNS disorders.

7. Singh M, Sumien N, Kyser C, Simpkins JW: Estrogens and progesterone as neuroprotectants: what animal models teach us. Front. Biosci. 13, 1083–1089 (2008).

8. Stein DG, Hurn PD: Effects of sex steroids on damaged neural systems. In: Hormones, Brain and Behavior. 2nd Edition. Pfaff DW, Arnold AP, Etgen AM et al. (Eds). Elsevier, Oxford, UK, 2223–2258 (2009).

n Recent comprehensive review comparing the uses of progesterone and estrogen in the treatment of TBI and stroke.

9. Stein DG: Progesterone exerts neuroprotective effects after brain injury. Brain Res. Rev. 57, 386–397 (2008).

10. Stein DG, Wright DW, Kellermann AL: Does progesterone have neuroprotective properties? Ann. Emerg. Med. 51, 164–172 (2008).

n Written for physicians wishing to learn more about progesterone in the treatment of TBI. This review includes a very brief overview of mechanisms of action.

11. Attella MJ, Nattinville A, Stein DG: Hormonal state affects recovery from frontal cortex lesions in adult female rats. Behav. Neural Biol. 48, 352–367 (1987).

12. Gibson CL, Gray LJ, Bath PM, Murphy SP: Progesterone for the treatment of experimental brain injury; a systematic review. Brain 131, 318–328 (2008).

13. Vandromme M, Melton SM, Kerby JD: Progesterone in traumatic brain injury: time to move on to Phase III trials. Crit. Care 12, 153 (2008).

14. Zygun DA, Kortbeek JB, Fick GH, Laupland KB, Doig CJ: Non-neurologic organ dysfunction in severe traumatic brain injury. Crit. Care Med. 33, 654–660 (2005).

15. Cai W, Zhu Y, Furuya K et al.: Two different molecular mechanisms underlying progesterone neuroprotection against ischemic brain damage. Neuropharmacology 55, 127–138 (2008).

16. Behera MA, Dai Q, Garde R et al.: Progesterone stimulates mitochondrial activity with subsequent inhibition of apoptosis in MCF-10A benign breast epithelial cells. Am. J. Physiol. Endocrinol. Metab. 297, E1089–E1096 (2009).

17. Chen G, Shi J, Ding Y, Yin H, Hang C: Progesterone prevents traumatic brain injury-induced intestinal nuclear factor-kb activation and proinflammatory cytokines expression in male rats. Med. Inflamm. 2007, 93431 (2007).

18. Hu Z, Li Y, Fang M, Wai MS, Yew DT: Exogenous progesterone: a potential therapeutic candidate in CNS injury and neurodegeneration. Curr. Med. Chem. 16, 1418–1425 (2009).

19. Sayeed I, Wali B, Stein DG: Progesterone inhibits ischemic brain injury in a rat model of permanent middle cerebral artery occlusion. Restor. Neurol. Neurosci. 25, 151–159 (2007).

20. Mani SK: Signaling mechanisms in progesterone–neurotransmitter interactions. Neuroscience 138, 773–781 (2006).

21. Mani SK, Portillo W, Reyna A: Steroid hormone action in the brain: cross-talk between signalling pathways. J. Neuroendocrinol. 21, 243–247 (2009).

22. Labombarda F, Gonzalez SL, Deniselle MC et al.: Effects of injury and progesterone treatment on progesterone receptor and progesterone binding protein 25-Dx expression in the rat spinal cord. J. Neurochem. 87, 902–913 (2003).

23. Sayeed I, Parvez S, Winkler-Stuck K et al.: Patch clamp reveals powerful blockade of the mitochondrial permeability transition pore by the D2-receptor agonist pramipexole. FASEB J. 20, 556–558 (2006).

24. Ziegler E, Bodusch M, Song Y et al.: Interaction of androsterone and progesterone with inhibitory ligand-gated ion channels: a patch clamp study. Naunyn Schmiedebergs Arch. Pharmacol. 380, 277–291 (2009).

25. Djebaili M, Guo Q, Pettus EH, Hoffman SW, Stein DG: The neurosteroids progesterone and allopregnanolone reduce cell death, gliosis, and functional deficits after traumatic brain injury in rats. J. Neurotrauma 22, 106–118 (2005).

26. Yao XL, Liu J, Lee E, Ling GS, McCabe JT: Progesterone differentially regulates pro- and anti-apoptotic gene expression in cerebral cortex following traumatic brain injury in rats. J. Neurotrauma 22, 656–668 (2005).

27. Wise PM: Estrogen therapy: does it help or hurt the adult and aging brain? Insights derived from animal models. Neuroscience 138, 831–835 (2006).

28. Guerra-Araiza C, Coyoy-Salgado A, Camacho-Arroyo I: Sex differences in the regulation of progesterone receptor isoforms expression in the rat brain. Brain Res. Bull. 59, 105–109 (2002).

29. You JM, Yun SJ, Nam KN et al.: Mechanism of glucocorticoid-induced oxidative stress in rat hippocampal slice cultures. Can. J. Physiol. Pharmacol. 87, 440–447 (2009).

30. Robertson CL, Puskar A, Hoffman GE et al.: Physiologic progesterone reduces mitochondrial dysfunction and hippocampal cell loss after traumatic brain injury in female rats. Exp. Neurol. 197, 235–243 (2006).

31. Roglio I, Bianchi R, Gotti S et al.: Neuroprotective effects of dihydroprogesterone and progesterone in an experimental model of nerve crush injury. Neuroscience 155, 673–685 (2008).

32. Garay L, Gonzalez Deniselle MC, Gierman L et al.: Steroid protection in the experimental autoimmune encephalomyelitis model of multiple sclerosis. Neuroimmunomodulation 15, 76–83 (2008).



33. De Nicola AF, Labombarda F, Deniselle MC et al.: Progesterone neuroprotection in traumatic CNS injury and motoneuron degeneration. Front. Neuroendocrinol. 30, 173–187 (2009).

34. Henderson LP: Steroid modulation of GABAA

receptor-mediated transmission in the hypothalamus: effects on reproductive function. Neuropharmacology 52, 1439–1453 (2007).

35. Pan DS, Liu WG, Yang XF, Cao F: Inhibitory effect of progesterone on inflammatory factors after experimental traumatic brain injury. Biomed. Environ. Sci. 20, 432–438 (2007).

36. Chen G, Shi J, Jin W et al.: Progesterone administration modulates TLRs/NF-kb signaling pathway in rat brain after cortical contusion. Ann. Clin. Lab. Sci. 38, 65–74 (2008).

37. Chen G, Shi JX, Qi M, Wang HX, Hang CH: Effects of progesterone on intestinal inflammatory response, mucosa structure alterations, and apoptosis following traumatic brain injury in male rats. J. Surg. Res. 147, 92–98 (2008).

38. Chen J, Chopp M, Li Y: Neuroprotective effects of progesterone after transient middle cerebral artery occlusion in rat. J. Neurol. Sci. 171, 24–30 (1999).

39. Rhodes ME, Frye CA: Actions at GABAA

receptors in the hippocampus may mediate some antiseizure effects of progestins. Epilepsy Behav. 6, 320–327 (2005).

40. Quadros PS, Pfau JL, Wagner CK: Distribution of progesterone receptor immunoreactivity in the fetal and neonatal rat forebrain. J. Comp. Neurol. 504, 42–56 (2007).

41. Moorthy K, Sharma D, Basir SF, Baquer NZ: Administration of estradiol and progesterone modulate the activities of antioxidant enzyme and aminotransferases in naturally menopausal rats. Exp. Gerontol. 40, 295–302 (2005).

42. Cutler SM, Cekic M, Miller DM et al.: Progesterone improves acute recovery after traumatic brain injury in the aged rat. J. Neurotrauma 24, 1475–1486 (2007).

43. Guo Q, Sayeed I, Baronne LM et al.: Progesterone administration modulates AQP4 expression and edema after traumatic brain injury in male rats. Exp. Neurol. 198, 469–478 (2006).

44. Vanlandingham JW, Cutler SM, Virmani S et al.: The enantiomer of progesterone acts as a molecular neuroprotectant after traumatic brain injury. Neuropharmacology 51, 1078–1085 (2006).

45. Charalampopoulos I, Remboutsika E, Margioris AN, Gravanis A: Neurosteroids as modulators of neurogenesis and neuronal survival. Trends Endocrinol. Metab. 19, 300–307 (2008).

46. Reddy DS, O’Malley BW, Rogawski MA: Anxiolytic activity of progesterone in progesterone receptor knockout mice. Neuropharmacology 48, 14–24 (2005).

47. Reddy DS, Apanites LA: Anesthetic effects of progesterone are undiminished in progesterone receptor knockout mice. Brain Res. 1033, 96–101 (2005).

48. Zheng P: Neuroactive steroid regulation of neurotransmitter release in the CNS: action, mechanism and possible significance. Prog. Neurobiol. 89(2), 134–152 (2009).

49. Akk G, Covey DF, Evers AS et al.: Mechanisms of neurosteroid interactions with GABA

A receptors. Pharmacol. Ther. 116,

35–57 (2007).

50. Covey DF: ent-Steroids: novel tools for studies of signaling pathways. Steroids 74, 577–585 (2009).

51. Jones LA, Anthony JP, Henriquez FL et al.: Toll-like receptor-4-mediated macrophage activation is differentially regulated by progesterone via the glucocorticoid and progesterone receptors. Immunology 125, 59–69 (2008).

52. Hwang JY, Duncan RS, Madry C, Singh M, Koulen P: Progesterone potentiates calcium release through IP3 receptors by an Akt-mediated mechanism in hippocampal neurons. Cell Calcium 45, 233–242 (2009).

53. Viero C, Dayanithi G: Neurosteroids are excitatory in supraoptic neurons but inhibitory in the peripheral nervous system: it is all about oxytocin and progesterone receptors. Prog. Brain Res. 170, 177–192 (2008).

54. Sakamoto H, Ukena K, Kawata M, Tsutsui K: Expression, localization and possible actions of 25-Dx, a membrane-associated putative progesterone-binding protein, in the developing Purkinje cell of the cerebellum: a new insight into the biosynthesis, metabolism and multiple actions of progesterone as a neurosteroid. Cerebellum 7, 18–25 (2008).

55. Guennoun R, Meffre D, Labombarda F et al.: The membrane-associated progesterone-binding protein 25-Dx: expression, cellular localization and up-regulation after brain and spinal cord injuries. Brain Res. Rev. 57, 493–505 (2008).

56. Quadros PS, Schlueter LJ, Wagner CK: Distribution of progesterone receptor immunoreactivity in the midbrain and hindbrain of postnatal rats. Dev. Neurobiol. 68, 1378–1390 (2008).

57. Zhu Y, Hanna RN, Schaaf MJ, Spaink HP, Thomas P: Candidates for membrane progestin receptors – past approaches and future challenges. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 148, 381–389 (2008).

58. Mani S: Progestin receptor subtypes in the brain: the known and the unknown. Endocrinology 149, 2750–2756 (2008).

59. McArthur DL, Chute DJ, Villablanca JP: Moderate and severe traumatic brain injury: epidemiologic, imaging and neuropathologic perspectives. Brain Pathol. 14, 185–194 (2004).

60. Onyszchuk G, He YY, Berman NE, Brooks WM: Detrimental effects of aging on outcome from traumatic brain injury: a behavioral, magnetic resonance imaging, and histological study in mice. J. Neurotrauma 25, 153–171 (2008).

61. Holick MF, Chen TC: Vitamin D deficiency: a worldwide problem with health consequences. Am. J. Clin. Nutr. 87, S1080–S1086 (2008).

n Brief review of the importance of considering the role of vitamin D in health and disease.

62. Grant WB: Epidemiology of disease risks in relation to vitamin D insufficiency. Prog. Biophys. Mol. Biol. 92, 65–79 (2006).

63. Corino A, D’Amelio P, Gancia R et al.: Hypovitaminosis D in internal medicine inpatients. Calcif. Tissue Int. 80, 76–80 (2007).

64. Chatfield SM, Brand C, Ebeling PR, Russell DM: Vitamin D deficiency in general medical inpatients in summer and winter. Intern. Med. J. 37, 377–382 (2007).

65. Cekic M, Cutler SM, Vanlandingham JW, Stein DG: Vitamin D deficiency reduces the benefits of progesterone treatment after brain injury in aged rats. Neurobiol. Aging DOI: 10.1016/j.neurobiolaging.2009.04.017 (2009) (Epub ahead of print).

n One of the first papers to show an empirical relationship between vitamin D deficiency and the outcome of TBI in aged rats. It also demonstrates that the deficiency can alter the benefits of post-injury progesterone treatment.

66. Pape HC, Tsukamoto T, Kobbe P et al.: Assessment of the clinical course with inflammatory parameters. Injury 38, 1358–1364 (2007).

67. Margulies S, Hicks R: Combination therapies for traumatic brain injury: prospective considerations. J. Neurotrauma 26, 925–939 (2009).



68. Cekic M, Sayeed I, Stein DG: Combination treatment with progesterone and vitamin D hormone may be more effective than monotherapy for nervous system injury and disease. Front. Neuroendocrinol. 30, 158–172 (2009).

nn One of the first comprehensive reviews on combinatorial therapy using both vitamin D and progesterone to treat TBI.

69. Atif F, Sayeed I, Ishrat T, Stein DG: Progesterone with vitamin D affords better neuroprotection against excitotoxicity in cultured cortical neurons than progesterone alone. Mol. Med. 15(9–10), 328–336 (2009).

70. Goss CW, Hoffman SW, Stein DG: Behavioral effects and anatomic correlates after brain injury: a progesterone dose–response study. Pharmacol. Biochem. Behav. 76, 231–242 (2003).

71. Mizwicki MT, Norman AW: The vitamin D sterol-vitamin D receptor ensemble model offers unique insights into both genomic and rapid-response signaling. Sci. Signal. 2(75), re4 (2009).

72. Eloranta JJ, Hiller C, Hausler S, Stieger B, Kullak-Ublick GA: Vitamin D3 and its nuclear receptor increase the expression and

activity of the human proton-coupled folate transporter. Mol Pharmacol. 76(5), 1062–1071 (2009).

73. Fernandes de Abreu DA, Eyles D, Féron F: Vitamin D, a neuro-immunomodulator: implications for neurodegenerative and autoimmune diseases. Psychoneuroendocrinology DOI: 10.1016/j.psyneuen.2009.05.023 (2009) (Epub ahead of print).

74. Samuel S, Sitrin MD: Vitamin D’s role in cell proliferation and differentiation. Nutr. Rev. 66, S116–S124 (2008).

75. Boyan BD, Schwartz Z: 1,25-Dihydroxy vitamin D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60-activated matrix vesicle matrix metalloproteinases. Cells Tissues Organs 189, 70–74 (2009).

76. Christakos S, Dhawan P, Benn B et al.: Vitamin D: molecular mechanism of action. Ann. NY Acad. Sci. 1116, 340–348 (2007).

Websites101. Ippolito CJ: Battlefield TBI: blast

and aftermath. www.psychiatrictimes.com/display/article/10168/56916 (Accessed October 2009)

102. Centers for Disease Control: What is traumatic brain injury. www.cdc.gov/ncipc/tbi/TBI.htm (Accessed September 2009)

103. ClinicalTrials.gov http://clinicaltrials.gov/ct2/show/NCT00822900?term=progesterone,+TBI&rank=1

Affiliationsn Milos Cekic, MA

Department of Emergency Medicine, Emory University School of Medicine, Atlanta, Georgia, USA Tel.: +1 404 712 2540 Fax: +1 404 727 2388 [email protected]

n Donald G Stein, PhD Clinic B, Suite 5100, 1365B Clifton Road NE, Emory University, Atlanta, GA 30322, USA Tel.: +1 404 712 2540 Fax: +1 404 727 2388 [email protected]

409Regen. Med. (2014) 9(4), 409–412 ISSN 1746-0751

part of

Editorial

10.2217/RME.14.32 © 2014 Future Medicine Ltd

Regen. Med.

Editorial9

4

2014

Keywords: combination therapy • granulocyte-colony stimulating factor • human umbilical cord blood • traumatic brain injury

Traumatic brain injury (TBI) is a major pub-lic health problem associated with death and permanent disability worldwide [1]. Both acute and chronic symptoms accompany TBI, with survivors suffering from progressive post-TBI pathological manifestations such as neuroinflammation coupled with behavioral dysfunctions including sensory motor defi-cits, learning and memory impairments and a range of neuropsychiatric symptoms includ-ing anxiety, depression and aggression [2,3]. At present, there is significant unmet need for clinically efficacious therapies for TBI [4].

Stem cell transplantation has been shown to be an effective regenerative therapy for functional and physiological improvement in animal models of brain disorders [5]. Among various types of stem cells, adult stem cells are of interest as they circumvent ethical and moral problems, as well as tera-togenic and oncogenic risks usually associ-ated with transplantation of embryonal- or fetal-derived stem cells [6]. In particular, a number of groups have focused on the potential of human umbilical cord blood (hUCB)-derived stem cells as a graft source for various intractable neurological disorders (e.g., stroke, Parkinson’s disease and Hun-tington’s disease, among others; for review see [6]). Moreover, clinical trials have been performed to determine the efficacy of hUCB stem cells in cerebral palsy, inborn metabolic disorders and stroke (for review see [7]).

hUCB stem cells have also been used experimentally in animal models of TBI. In some studies, transplanted cells derived from the mononuclear fraction of hUCB

have been shown to confer neuroprotection by decreasing inflammation and brain tissue loss, promoting neurogenesis and rescue of neurological dysfunctions [8,9]. However, an issue associated with hUCB transplantation, as with utilizing other cell types, involves limited regenerative capacity of transplanted stem cells due to the inhospitable micro-environment of the injured brain [10]. There-fore, a combination treatment that creates a conducive host–tissue microenvironment for the transplanted stem cells may improve the therapeutic outcome.

The granulocyte-colony stimulating fac-tor (G-CSF), an essential member of the hematopoietic growth factor family, has been shown to be neuroprotective in animal mod-els of stroke [11] and it also improved cogni-tion, reduced central and peripheral inflam-mation and enhanced neurogenesis in models of Alzheimer’s disease [12]. The safety and efficacy of G-CSF for acute ischemic stroke has already been explored in clinical studies [13,14]. However, G-CSF treatment in animal models of TBI produced discrepant results. While some studies reported improvement of TBI-associated behavioral and histological impairments, others found minimal effects of G-CSF on functional and neurological outcomes in animals subjected to TBI [15,16].

In a recent study, Acosta et al. [17] tested the putative benefits offered by combina-tion therapy in TBI by investigating whether there is enhanced behavioral and histological improvement after transplantation of hUCB and co-administration of G-CSF in a con-trolled cortical impact model of moderate

Umbilical cord blood cell and granulocyte-colony stimulating factor: combination therapy for traumatic brain injury

Ike de la PeñaDepartment of Neurosurgery & Brain

Repair, Center of Excellence for Aging

& Brain Repair, University of South

Florida Morsani College of Medicine,

Tampa, FL 33612, USA

Paul R SanbergDepartment of Neurosurgery & Brain





Sandra AcostaDepartment of Neurosurgery & Brain





Shinn-Zong LinCenter for Neuropsychiatry, China

Medical University & Hospital, Taichung,

Taiwan, PR China

Cesar V BorlonganAuthor for correspondence:

Department of Neurosurgery & Brain





Tel.: +1 813 974 3988

Fax: +1 813 974 3078

[email protected]



Editorial de la Peña, Sanberg, Acosta, Lin & Borlongan

TBI in adult rats. Their study revealed that while monotherapy with hUCB or G-CSF promoted behav-ioral recovery, combined therapy of hUCB plus G-CSF enhanced further functional improvement exerted by the latter treatment regimen. Moreover, the effects of combination therapy were longer lasting than those exerted by hUCB transplantation or G-CSF adminis-tration alone. Complementary brain-repair processes distinctly or mutually solicited by these two thera-pies could have mediated improved functional recov-ery exerted by combination treatment of hUCB plus G-CSF. Endogenous stem cells mobilized by G-CSF from the bone marrow to the peripheral blood (and ultimately to the site of injury) [11–14], growth factors secreted by hUCB grafts and the potential graft–host integration that leads to reconstruction of synaptic cir-cuitry [18] altogether could have facilitated the regener-ative mechanisms exerted by the combination therapy, but not by monotherapy. Further studies are needed to clarify this multipronged mechanism of action by the combination hUCB plus G-CSF therapy.

Acosta et al. also performed immunohistochemical staining with OX-6, which labels MHCII+ cells (puta-tively activated microglia), to determine the extent to which combination therapy of hUCB plus G-CSF ameliorated TBI-induced neuroinflammation in gray and white matter areas [17]. In parallel to the improved behavioral recovery with combination therapy, the investigators found that combined hUCB plus G-CSF treatment resulted in more profound reduction of TBI-induced upregulation of MHCII+ cells in the cortex, striatum, thalamus, subventricular zone and the den-tate gyrus of the hippocampus, compared with hUCB and/or G-CSF monotherapy. Moreover, whereas hUCB transplantation or G-CSF treatment alone sup-pressed activated MHCII+ cells in the corpus callosum and fornix, combined therapy of hUCB plus G-CSF decreased the number of MHCII+ cells not only in the corpus callosum and fornix, but also in the cerebral peduncle [17].

That combination therapy enhanced the therapeutic outcome in TBI begs the question whether synergistic effects were produced by hUCB plus G-CSF in attenu-ating TBI-induced impairment in endogenous neuro-genesis and hippocampal cell loss. In animal models of

TBI, as well as in preclinical stroke and aging studies, hUCB treatment has been shown to decrease inflam-mation and facilitate neurogenesis and angiogenesis [19,20]. In a similar fashion, treatment with G-CSF has been shown to modulate neurogenesis in TBI, as well as in other neurodegenerative disorders (e.g., hypoxic injury and Alzheimer’s disease, among others) [12]. The findings from the studies of Acosta et al. showed that reduction in neuroinflammation exerted by hUCB plus G-CSF therapy coincided with elevated neurogenesis in dentate gyrus and subventricular zone while increas-ing the survival of CA3 neurons in TBI rats. Alto-gether, the results from the above-mentioned studies indicate that combined therapy of hUCB plus G-CSF synergistically dampened TBI-induced neuroinflam-mation while significantly enhancing endogenous neurogenesis and reducing hippocampal cell loss [17].

A complementary interaction between hUCB and G-CSF may also explain the widespread effects of this combination therapy in diverse brain regions. As men-tioned above, G-CSF displays the capacity to mobi-lize stem cells from the bone marrow to the periph-eral blood, and the mobilized cells have been shown to infiltrate injured tissues promoting self-repair of neurons [11–14]. Additionally, G-CSF permeates the blood–brain barrier and acts upon neurons and glial cells through the G-CSF receptor [21]. Among the many beneficial effects of G-CSF receptor activation in neurons and glia include downregulation of proinflam-matory cytokines and increased neurogenesis [22,23]. Moreover, the combination of hUCB plus G-CSF may promote stemness maintenance, and under appropriate conditions, guide neural lineage commitment of hUCB [24]. On the other hand, mobilized bone marrow cells and hUCB cells may also exert their therapeutic ben-efits via a paracrine mechanism, in other words, trans-planted cells secrete trophic factors, growth factors, chemokines and immune-modulating cytokines to the injured milieu, in line with the concept of ‘bystander effects’ of transplanted stem cells [25,26]. In summary, a receptor-mediated transport mechanism coupled with paracrine effects of transplanted cells may underlie extensive influence of combination therapy of hUCB plus G-CSF in diverse brain regions as evidenced by enhanced anti-inflammatory, improved neurogenesis

“...a receptor-mediated transport mechanism coupled with paracrine

effects of transplanted cells may underlie extensive influence of combination therapy

of human umbilical cord blood plus granulocyte-colony stimulating factor in

diverse brain regions...”

“...combined therapy of human umbilical cord blood plus granulocyte-

colony stimulating factor synergistically dampened traumatic brain injury-induced

neuroinflammation while significantly enhancing endogenous neurogenesis and

reducing hippocampal cell loss.”


hUCB & G-CSF: combinatorial therapeutics for traumatic brain injury Editorial

and increased cell survival in TBI rats that received hUCB plus G-CSF.

Conclusion & future perspectiveIn the clinic, chronic TBI has been recently recognized as a progressive cell death process rather than an acute event, characterized by a worsening histopathology with limited therapeutic opportunities [27]. The study of Acosta et al. demonstrated long-lasting recovery of motor functions and more robust attenuation of neu-roinflammation accompanied by enhanced neuro-genesis and profound rescue against neuronal cell death in TBI rats given hUCB plus G-CSF. This study showed how stand-alone therapies (i.e., hUCB and G-SCF) could overcome their therapeutic limitations in chronic TBI when synergy is accomplished through combination therapy. That TBI-associated cell death cascades require multipronged regenerative processes highlights the need for combination therapy targeting different cell death or survival pathways. Such multi-ple biologic therapeutic approaches open new research directions and clinical applications for TBI. Consider-ing that hUCB and G-CSF present with solid safety

and efficacy profiles as stand-alone therapies, their combination therapy appears indicated for limited clinical trials in TBI.

Financial & competing interests disclosure The study by Acosta et al. was funded by the Department of

Defense W81XWH-11-1-0634, the University of South Florida

Signature Interdisciplinary Program in Neuroscience funds, the

University of South Florida and Veterans Administration Reinte-

gration Funds and the University of South Florida Neuroscience

Collaborative Program. PR Sanberg and CV Borlongan serve as

consultant and founder, respectively, of Saneron CCEL Thera-

peutics, Inc. CV Borlongan is funded by the National Institutes

of Health (1R01NS071956-01A1) and James and Esther King

Biomedical Research Foundation (1KG01-33966). The funders

had no role in study design, data collection and analysis, deci-

sion to publish or preparation of the manuscript. The authors

have no other relevant affiliations or financial involvement

with any organization or entity with a financial interest in or

financial conflict with the subject matter or materials discussed

in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this

manuscript.

References1 Faul M, Xu L, Wald MM, Coronado VG. Traumatic Brain

Injury in the United States: Emergency Department Visits, Hospitalizations and Deaths. Centers for Disease Control and Prevention, National Center for Injury Prevention and Control, Atlanta, GA, USA (2010).

2 Azouvi P, Vallat-Azouvi C, Belmont A. Cognitive deficits after traumatic coma. Prog. Brain Res. 177, 89–110 (2009).

3 Wong D, Dahm J, Ponsford J. Factor structure of the depression anxiety stress scales in individuals with traumatic brain injury. Brain Inj. 27, 1377–1382 (2013).

4 Kaneko Y, Tajiri N, Yu S et al. Nestin overexpression precedes caspase-3 upregulation in rats exposed to controlled cortical impact traumatic brain injury. Cell Med. 4(2), 55–63 (2012).

5 Huang HY, Chen L, Sanberg PR. Clinical achievements, obstacles, falsehoods, and future directions of cell-based neurorestoratology. Cell Transplant. 21(1), S3–S11 (2012).

6 Sanberg PR, Eve DJ, Metcalf C et al. Advantages and challenges of alternative sources of adult-derived stem cells for brain repair in stroke. Prog. Brain Res. 201, 99–117 (2012).

7 Ilic D, Miere C, Lazic E. Umbilical cord blood cells: clinical trials in non-hematological disorders. Br. Med. Bull. 102, 43–57 (2012).

8 Mahmood A, Lu D, Qu C et al. Long-term recovery after bone marrow stromal cell treatment of traumatic brain injury in rats. J. Neurosurg. 104, 272–277 (2006).

9 Tajiri N, Acosta S, Glover LE et al. Intravenous grafts of amniotic fluid-derived stem cells induce endogenous cell proliferation and attenuate behavioral deficits in ischemic stroke rats. PLoS ONE 7, e43779 (2012).

10 Walker PA, Shah SK, Jimenez F, Aroom KR, Harting MT, Cox CS Jr. Bone marrow-derived stromal cell therapy for traumatic brain injury is neuroprotective via stimulation of non-neurologic organ systems. Surgery 152, 790–793 (2012).

11 Shyu WC, Lin SZ, Yang HI et al. Functional recovery of stroke rats induced by granulocyte colony-stimulating factor-stimulated stem cells. Circulation 110(13), 1847–1854 (2004).

12 Sanchez-Ramos J, Song S, Sava V et al. Granulocyte colony stimulating factor decreases brain amyloid burden and reverses cognitive impairment in Alzheimer’s mice. Neuroscience 163, 55–72 (2009).

13 England TJ, Abaei M, Auer DP et al. Granulocyte-colony stimulating factor for mobilizing bone marrow stem cells in subacute stroke: the stem cell trial of recovery enhancement after stroke 2 randomized controlled trial. Stroke 43, 405–411 (2012).

14 Shyu WC, Lin SZ, Lee CC et al. Granulocyte colony-stimulating factor for acute ischemic stroke: a randomized controlled trial. CMAJ 174(7), 927–933 (2006).

15 Sakowitz OW, Schardt C, Neher M et al. Granulocyte colony-stimulating factor does not affect contusion size, brain edema or cerebrospinal fluid glutamate concentrations in rats following controlled cortical impact. Acta Neurochir. Suppl. 96, 139–143 (2006).

16 Yang DY, Chen YJ, Wang MF et al. Granulocyte colony-stimulating factor enhances cellular proliferation and motor function recovery on rats subjected to traumatic brain injury. Neurol. Res. 32(10), 1041–1049 (2010).

17 Acosta S, Tajiri N, Shinozuka K et al. Combination therapy of human umbilical cord blood cells and granulocyte colony stimulating factor reduces histopathological and motor


Editorial de la Peña, Sanberg, Acosta, Lin & Borlongan

impairments in an experimental model of chronic traumatic brain injury. PLoS ONE 9(3), e90953 (2014).

18 Willing AE, Vendrame M, Mallery J et al. Mobilized peripheral blood cells administered intravenously produce functional recovery in stroke. Cell Transplant. 12, 449–454 (2003).

19 Iskander A, Knight RA, Zhang ZG et al. Intravenous administration of human umbilical cord blood-derived AC133+ endothelial progenitor cells in rat stroke model reduces infarct volume: magnetic resonance imaging and histological findings. Stem Cells Transl. Med. 2, 703–714 (2013).

20 Shahaduzzaman M, Golden JE, Green S et al. A single administration of human umbilical cord blood T cells produces long-lasting effects in the aging hippocampus. Age (Dordr.) 35, 2071– 2087 (2013).

21 Zhao LR, Piao CS, Murikinati SR et al. The role of stem cell factor and granulocyte-colony stimulating factor in treatment of stroke. Recent Pat. CNS Drug Discov. 8, 2–12 (2013).

22 Schneider A, Kruger C, Steigleder T et al. The hematopoietic factor G-CSFs is a neuronal ligand that counteracts programmed cell death and drives neurogenesis. J. Clin. Invest. 115, 2083–2098 (2005).

23 Toth ZE, Leker RR, Shahar T et al. The combination of granulocyte colony-stimulating factor and stem cell factor significantly increases the number of bone marrow-derived endothelial cells in brains of mice following cerebral ischemia. Blood 111, 5544–5552 (2008).

24 Stachura DL, Svoboda O, Campbell CA et al. The zebrafish granulocyte colony stimulating factors (GCSFs): two paralogous cytokines and their roles in hematopoietic development and maintenance. Blood 122(24), 3918–3928 (2013).

25 Borlongan CV. Bone marrow stem cell mobilization in stroke: a ‘bonehead’ may be good after all! Leukemia 25, 1674–1686 (2011).

26 Yang M, Wei X, Li J et al. Changes in host blood factors and brain glia accompanying the functional recovery after systemic administration of bone marrow stem cells in ischemic stroke rats. Cell Transplant. 19, 1073–1084 (2010).

27 Lewis FD, Horn GJ. Traumatic brain injury: analysis of functional deficits and post-hospital rehabilitation outcomes. J. Spec. Oper. Med. 13, 56–61 (2013).

brain injury - future medicine...future cience rou 919 stem cell therapies for traumatic brain...

Documents