Naoki Tajiri1, Yuji Kaneko1, Kazutaka Shinozuka1, Hiroto Ishikawa1 et.al Plos One, 8(9)Sept 2013

Sandeep SatapathyRoll-10079

Stem Cell Recruitment of Newly Formed Host Cells via a Successful Seduction? Filling the Gap between Neurogenic Niche and Injured Brain Site

Exploring the Neurogenic Niche-

• The Sub-Ventricular Zone (SVZ) and the Sub Granular Zone /Dentate Gyrus (DG) of the Hippocampus of the brain – active sites of Neuronal Stem Cells(NSCs) in the brain.

• However, in ischemic conditions the cortical region of brain has been shown to be actively involved in formation of new neurons from these NSCs.

"Neural Stem Cells - New Perspectives”, DOI: 10.5772/55426

So, in the cortex, where does these NSCs come from????

Hypothesis:

Probably the cortex in a post injury condition, is facilitated to receive the NSCs from the active stem niche like , SVZ and DG of hippocampus, in order to compensate for the neuronal damage to regain the impaired function??

If, this happens then; how does this migration takes place in the brain???provided there are other cerebral tissues, which might pose to be potential barrier in this long distance migration of NSCs.

Is there any other mediating pathway or factor that acts as a bridge or the so called BIOBRIDGE , for this seduction of NSCs to happen????

Does the migratory pathway also manifest certain Extra cellular interacting molecules, which in a concerted process is allowing this migration to occur.

Ramification of Ideas:Classically stem cells were looked as

• Source of Cell Replacement• Bystander effects trophic factor secretion at localized tissues.

However, in present times, Stem cells have made a leap

• Into translational biology and applications of regenerative medicines with approaches of modulation the Extracellular matrix biology.

So, this paper for the first time establishes a Stem cell mediated bio ridge between the SVG /DG, and the site of injury, i.e. the cortical region and existence of such a connector is characterized by the increase in expression and activity of extracellular metalloproteinase (MMP-9).

Traffic Regulator:

NSCs from SVZ/DG

Cortical Tissues of brain-site of injury/stroke

Intracerebral Transplanted Stem cells

Features:

• High level of extracellular metalloproteinases following a Intracerebral Stem Cell implantation. The engrafted stem cell had a very short persistence time.***

• Decreases host cell death and proliferation and formation of new host cells.

• Endogenous repair pathway onset, post a stem cell bio bridge formation and regain of impaired motor and neurological functions.

Why low persistence time of engrafts not an issue??

Prior to the start of the preliminary experiments, it was expected that the Intracerebral infused MSCs would have very less retention potential at the site of peri cortical lesions or the migratory exodus path from the SVZ/DG to the Cortex in ischemic rats.

This was because of Hatting Mt, et.al(2009) who proved that in very short time period, around a 20-30 days post MSCs infusion the stem cells permeate into the pulmonary vasculature and start circulating in the arterial blood system.

This effect was termed as “PULMONARY FIRST PASS EFFECT”.

Findings:

Post 1 month graft survival: 0.6%

Post 3 months graft survival :0.16%

Procedures:

1. Control Animal Parameterization:

Prior to impacted brain injury, the animals were checked for baseline motor and neurological functions and this was classified in terms of,

• EBST: Elevated body swing test,10 degrees shift from the central bodyline in a hanging condition.

• Rotorod Maintenance Potential: The time period upto which the rats could keep themselves stable on a fixed frequency rotating rod-Motor Coordination Index.

• Bederson’s Neurological Score: Based on the observable phenotypes like front limb retraction post lateral displacement of 2-3 cms, beam walking ability under a focused beam. These were scored from 0 sec (immediate response) to 3 sec (delayed response)

2. Traumatic Brain Injury Surgery (TBI):

The anesthetized rat animal models ,underwent craniectomy and using a precise metal impactor, the cortical region of the brain was given a uniform impact injury.

3.Graft Procedures:

The grafting of the messencymal stem cells (MSCs) were done at Intracerebral locations(median cortex region and peri-cortex lesions) , days 7 post TBI, under anesthetized conditions of the rat model. Around 105 cells were grafted for 3 ul per deposit and three rounds were repeated of the same. The grafting was done post checking of cross reactivity with monoclonal human specific antibody (HuNu).Animals were left at 37°C for recovery and survival.

4.Cell Survival Analyses: Using high power field microscope, from a cryo preserved section of the cortical region of the brain, the number of surviving host cells were calculated in the peri cortex lesion area.

Histology:

1.Analyses of brain damage :

This was done staining the 4 coronal tissue sections of the dissected brain using Hemotoxylin(H) & Eosin (E) stain. Together with this data and from the calculated date of cell survivability in peri-cortical lesion area, the span of tissue damage and the new host cell proliferation was accounted for post stem cell transplantation. Using Abercrombie's formula the calculation of per section was summed up for the entire brain.

2.Additional Immunohistochemical Analyses: Apart from analyzing the host cell survivability and brain damage, to substantiate for the stem cell bridge the following Immunohistochemical markers were also analyzed:

• For Host cell proliferation : Ki67 Marker• Migration of NSCs from SVZ/DG to Peri cortex/cortex: Double cortin /DCX

marker.• Immature neural marker :Nestin

Zymography:To study the manifestation of unregulated levels of extracellular metalloproteinases (MMP-9) , Zymography was done with gelatin based SDS PAGE. The density of the bands forming clear strands against the darkly stained undigested gel (stained using coomassie brilliant blue) helps to quantify the expressional levels/activity of a hydrolytic enzyme like MMP-9.Recombinant enzyme was used as control strand in each gel.

Autoradiography:

Because of the metals present in the enzyme posing for radioactive properties, the tissue connecting the SVZ/DG to Cortex was cryodissected and the membrane was blocked and further the MMP-9 was subjected to anti-MMP 9 antibody (mouse monoclonal),which later was amplified using goat anti mouse antibody ,conjugated with Horse radish peroxidase for Immunofluorescence and autoradiography assay.

Cell Migration Assay:

The Boyden chamber loaded with PRNCs (primary rat neuronal Cells),was placed on a 24 well plate with confluent levels of MSCs (105 cells/well) and starved with serum free DMEM/F-12 along with

Presence of cyclosporine A

Absence of cyclosporine A

Cyclosporine A is known to inhibit MMP-9

Results:

Figure 1. Behavioral tests (performed by two investigators blinded to the treatment condition throughout the study) were initially conducted at baseline (i.e., prior to brain insult) and revealed that all adult SD rats included in this study displayed normal behaviors (A, B, and C).

Figure 2. The bio bridge between SVZ and impacted cortex consists of highly proliferative, neutrally committed, and migratory cells.

Figure 3. At three months post-TBI, the brains from vehicle-infused animals displayed a disparate pattern of cell fate in that the newly formed Ki67 positive and nestin labeled cells were sequestered within the corpus callosum (A) and the SVZ (B) and only sporadic cells were able to reach the impacted cortex (A’ and B’), with likely resident DCX cells seen around the impacted cortex (C).

Figure 4. Laser-captured biobridge, corresponding to the brain tissue between SVZ and impacted cortex, expressed high levels of MMP-9 gelatinolytic activities at one month and three months post-TBI in animals transplanted with SB623 which were significantly higher than those TBI animals that received vehicle only or sham-operated animals (*p’s< 0.05 vs. vehicle or sham; Panel A).

Figure 5. After TBI, endogenous repair mechanisms commenced, but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A).

Shortcomings:

Specific concentration to the median cortex area and peri lesion cortex area, and thereby calculation of motor coordination index and neurological score might give rise to certain false positives, as the peripheral brain damage was ignored during baseline behavior parameterization.

Future Possibilities:

• In most adult tissues, it is seen that the stem cell niche is kept aloof from the active functioning cells and its recruitment follows a necessity of repair and regeneration.So,in diseases where this recruitment process fails, can be compensated for infused stem.

• Further the remodeling of host cells (into undifferentiated stem cells), at the site connecting the stem cell niche and the zone of their functional requirement ,can potential compensate for the need for external infusion strategies.

• Migration of host cells from their stem cell niche to their zone of active functional requirement, requires up regulation of MMPs and thus ECM proteins like MMPs, can act as potential stroke therapies.

• Ultimately we can have something called “ STEM CELL VACCINES”

Take Home:

Stem cells may serve as bio bridges expressing MMP profiles, that recapitulate the neurovascular unit abetting the transplant mediated host cell migration towards injured brain areas in affording functional recovery in TBI.

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