Journal of Neuroscience Research 44957-164 (1996)

Increased Expression of Brain-Derived Neurotrophic Factor But Not Neurotrophin-3 mRNA in Rat Brain After Cortical Impact Injury K. Yang, J.R. Perez-Polo, X.S. Mu, H.Q. Yan, J.J. Xue, Y. Iwamoto, S.J. Liu, C.E. Dixon, and R.L. Hayes Department of Neurosurgery, University of Texas Health Sciences Center at Houston, Houston (K.Y., X.S.M., H.Q.Y., J. J.X., Y . I . , S.J.L., C.E.D., R.L.H.), Department of Human Biochemistry and Genetics, University of Texas Medical Branch, Galveston (J.R.P.-P.), Texas

Levels of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) mRNA expression were measured in a rodent model of traumatic brain injury (TBI) following unilateral injury to the cerebral cor- tex. To obtain reliable data on the co-expression of neurotrophin genes, adjacent coronal sections from the same rat brains were hybridized in situ with BDNF and NT3 cRNA probes. BDNF mRNA in- creased at 1, 3, and 5 hr after unilateral cortical in- jury in the cortex ipsilateral to the injury site and bilaterally in the dorsal hippocampus. NT3 mRNA did not change significantly following injury. Our re- sults suggest that TBI produces rapid increases in BDNF mRNA expression in rat brain without changes in NT3 mRNA expression, a finding which differs from studies of ischemia and seizures. It is possible that increased levels of BDNF mRNA rather than NT3 are important components of pathophysio- logical responses to TBI.

Key words: gene expression, neurotrophin, BDNF, NT3, traumatic brain injury

o 1996 Wiley-Liss, h c .

INTRODUCTION Substantial progress has been made in understand-

ing components of the pathologic responses to traumatic brain injury (TBI), some of which could modulate neu- rotrophin expression. TBI produces widespread mem- brane depolarization and enhanced release of excitatory neurotransmitters (reviewed in Hayes et al., 1992; McIn- tosh, 1993) that could contribute to intracellular calcium increases (Fineman et al., 1993; Smith et al., 1993) and activation of calcium-dependent enzymes known to play an important role in signal transduction. Alterations in

signal transduction systems, including changes in protein kinase C (PKC) activity, have been reported after TBI (Yang et al., 1993; Prasad et al., 1994). The activation of immediate early genes (IEGs) also represents a poten- tially important interface between initial cellular re- sponses to central nervous system (CNS) injury and later, more enduring, pathologic results. Increases in c-fos mRNA (Yang et al., 1994) and protein (Phillips and Belardo, 1992) in the hippocampus have been re- ported after TBI. Increases in excitatory neurotransmit- ters and intracellular calcium levels, as well as enhanced protein kinase activity and expression of immediate early genes, may all be components of intracellular cascades necessary for increased neurotrophin gene expression af- ter CNS injury, such as the possible induction of NGF mRNA by c-fos after TBI in rats (Yang et al., 1995).

Brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3) are neurotrophins present in the rat brain (Hofer et al., 1990; Maisonpierre et al., 1990; Rocamora et al., 1992; Smith et al., 1995). Inter- estingly, the distributions of cells expressing BDNF and NT3 mRNA in the adult rat brain are unique for each factor. In the hippocampus, BDNF mRNA is expressed in the dentate g y m and CAl-CA4 regions, whereas NT3 mRNA is expressed in a more restrictive pattern, being distributed only in the dentate gyrus, CA2, and medial CAI regions (Rocamora et al., 1992; Smith et al., 1995). Outside of the hippocampus, higher levels of BDNF mRNA have been found in many regions of the

Received July 18, 1995; revised November 3, 1995; accepted Novem- ber 10, 1995. Address reprint requests to Ronald L. Hayes, Ph.D., Department of Neurosurgeq, University of Texas Health Science Center at Houston, 6431 Fannin Street, Suite 7.154, Houston, TX 77030.

0 1996 Wiley-Liss, Inc.

158 Yang et al.

brain, including neocortex, piriform cortex, and amygdala, whereas NT3 mRNA is not markedly ex- pressed in these areas (Hohn et al., 1990; Lindvall et al., 1992; Rocamora et al., 1992). Both neurotrophins, how- ever, produce a similar neurotrophic effect on some neu- rons, while retaining distinct characteristics in other re- spects (Lindsay et al., 1985; Maisonpierre et al., 1990; Anderson et al., 1990; Hohn et al., 1990; Hyman et al., 1991; Knusel et al., 1992). Both BDNF and NT3 effect neurite outgrowth (Davies et al., 1986; Maisonpierre et al., 1990) and the survival of neural crest-derived sen- sory neurons (Barde et al., 1980; Lindsay et al., 1985; Davies et al., 1986; Hohn et al., 1990). Further studies indicate that BDNF may be trophic for a wide range of different types of neurons (DiStefano et al., 1992; Ip et al., 1993; Smith et al., 1995). BDNF, but not NT3, increases survival of cholinergic neurons (Anderson et al., 1990; Ballarin et al., 1991; Dixon et al., 1991) and dopaminergic neurons from the substantia nigra (Hyman et al., 1991; Knusel et al., 1991). In contrast, NT3 is considered to be particularly important in embryogenesis and development of the hippocampus (Ernfors et al., 1990; Friedman et al., 1991; Collazo et al., 1992).

Several studies have examined the effects of CNS injuries on expression of BDNF and NT3. CNS insults associated with activation of glutamate transmission, in- creased intracellular calcium, and c-fos gene expression have demonstrated increased levels of BDNF gene ex- pression. Increased BDNF expression has been reported following ischemia (Lindvall et al., 1992; Takeda et al., 1993), seizures (Gall and Isackson, 1989; Isackson et al., 1991; Gall and Lauterborn, 1992; Rocamoraet al., 1992), hippocampal damage induced by kainic acid injection (Ballarin et al., 1991), insulin-induced hypoglycemic coma (Lindvall et al., 1992), glutamate-mediated spread- ing depression (Kokaia et al., 1993), and glucocorticoid- associated stress (Smith et al., 1995). Decreased NT3 mRNA expression has been reported following ischemia (Lindvall et al., 1992; Takeda et al., 1992), seizures (Ernfors et al., 1990; Isackson et al., 1991; Gall and Lauterborn, 1992; Rocamora et al., 1992), and insulin- induced hypoglycemic coma (Lindvall et al., 1992).

No studies have examined BDNF and NT3 mRNA expression following mechanical traumatic brain injury. Thus, we employed a controlled cortical impact rodent model of brain injury to investigate the co-expression of BDNF and NT3 in the rat brain. This model was devel- oped in our laboratory and is widely employed in other laboratories (Dixon et al., 1991). The model reproduces features of human TBI including cortical contusions and long-lasting neurological deficits (Dixon et al., 1991). We report that TBI produces rapid increases in BDNF mRNA expression in rat brain without changes in NT3 mRNA expression.

MATERIALS AND METHODS Production of Traumatic Brain Injury

Male Sprague-Dawley rats (250-350 g, Harlan Laboratories, Houston, TX) were initially anesthetized with 4% isoflurane and N,O/O, (2:l) in a vented anes- thesia chamber. Following endotracheal intubation, rats were mechanically ventilated with 2% isoflurane and se- cured in a stereotaxic frame. A midline incision was made, the soft tissues were reflected, and a circular sec- tion of skull 8 mm in diameter was removed from the right margin of the skull midway between the frontal and occipital sutures, 2 mm medial to the temporal ridge. The dura was left intact. This craniotomy exposed the injury site in all animals. Injury was produced by a con- trolled lateral cortical impact model described in detail by Dixon et al. (1991). The impact velocity was adjusted to 6.0 d s e c by controlled gas pressure and verified by a time-displacement curve that was measured by a linear variable differential transformer (LVDT) (Shaevitz Model 500 HR), and a PC-based data acquisition system (R. C. Electronics). Sham-injured rats were surgically prepared for injury but not injured.

All animal studies carefully conformed to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals from the U. S. Department of Health and Human Services, and were approved in advance by the University of Texas Health Sciences Center at Hous- ton Animal Welfare Committee.

Assessments of Transient Neurological Behavior Deficits

In order to ensure consistency of injury, transient behavioral suppression was assessed in injured and sham-injured rats employing a battery of tests routinely conducted in our laboratories (Dixon et al., 1991). As- sessments of transient behavioral changes were per- formed immediately after injury. Simple postural so- matomotor functions were assessed by measuring the duration of suppression of hind paw and tail flexion re- flexes. More complex postural somatomotor functions were assessed by recording the duration of suppression of head support, righting, and escape responses. Head support, a measure of nuchal muscle tone, was assessed by measuring the duration of suppression of the animal’s ability to support the weight of its head. The righting response was tested by placing the animal on its back and observing a spontaneous return to the upright position. The escape response was assessed by applying a pinch (0.8 kg/mm2) to the tail to elicit an organized locomotor response away from the noxious stimulus.

In Situ Hybridization We employed an in situ hybridization technique to

locate regions of possible changes in BDNF and NT3

Increased BDNF, Not NT3, mRNA in Injured Rat Brain 159

mRNA expression after injury. Importantly, hybridiza- tion of BDNF and NT3 mRNA employed adjacent sec- tions from the same rat. Five min, 30 min, 1 hr, 3 hr, and 5 hr after injury and sham injury (surgical preparation without injury: n = 3/group), rats were perfused intra- cardially with 120 ml saline at 40 mumin, followed by 200 ml of fixative A (0.8 g NaOH, 8 g paraformalde- hyde, and 1.64 g sodium acetate in 200 ml distilled H,O, pH 6.5) at 20 ml/min, and then fixative B (1.4 g NaOH, 14 g paraformaldehyde, and 13.35 g borax, pH 9.5). Rat brains were cryoprotected in 25% sucrose/fixative B overnight at 4°C. Coronal slices (15 pm) were prepared and mounted on poly-lysine-coated slides. The in situ hybridization technique employed is routinely used by our laboratory and has been previously described (Sim- mons et al., 1989; Yang et al., 1994). The samples were subjected to 0.001% proteinase K digestion at 37°C for 20 min, and then immersed in 0.1 M triethanolamine (TEA) with 0.25% acid anhydride for 10 min. Subse- quent dehydration was carried out in 50%, 70%, 95%, and 100% ethanol for 3 min each. Hybridization was performed with 33P-labeled cRNA probe ( lo7 cpdml ) overnight at 55°C. The cRNA probe was obtained from cDNA cloned in a pKS- vector using T7 or T3 RNA polymerase. After hybridization, the slides were washed sequentially in 2 X , 1 X , 0.2 X , and 0.1 X SSC, and dehydrated in 50%, 70%, 95%, and 100% ethanol for 3 min each. Brain slices were exposed to Kodak XAR-5 film for various time points at -70°C with intensifying screens.

Image Quantification and Statistical Analyses The levels of BDNF and NT3 mRNA expression in

rat brain after injury and sham injury procedures were quantified by computerized image analysis using NIH Image software (NIH Image 1.5 1, National Institute of Mental Health, Bethesda, MD) and a Macintosh com- puter (Apple Computer Inc., Cupertino, CA), using methods similar to those previously reported (Wetmore et al., 1990; Ballarin et al., 1991; Ernfors et al., 1991; Lindvall et al., 1992; Rocamora et al., 1992; Kokaia et al., 1993). Six different sections from 3 animals in each experimental paradigm were included in the analysis. In each section, optical densities were measured in the neo- cortex, dentate gyrus, and CA1-CA4. The optical den- sity of the corpus callosum was used to define back- ground hybridization, and this value was subtracted from each regional value within each section. Statistical anal- yses of changes in density levels between groups em- ployed a one-way analysis of variance (ANOVA). The differences were considered statistically significant if P 5 0.05. In this semi-quantitative analysis, results were expressed as the mean percentage of difference from mean sham injury control values, * standard error of the

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Fig. 1. Duration of postural somatomotor behavioral suppres- sion after controlled cortical impact injury in rats. The duration of these behavioral indices is consistent with previous reports of a controlled cortical impact injury. Data represent mean durations of suppression, 5 SE. Behavioral suppression was significantly increased in the injured group in all categories (P < 0.05). Statistical significance was calculated using an un- paired Student’s t-test. Sample size was n = 3 in the sham group and n = 15 in the injured group. Left Flex, Right Flex, Tail Flex: duration of suppression of flexion reflex to noxious pinch to left or right hind paw. Escape: duration of suppression of organized escape response to noxious tail pinch. Head Up: duration of suppression of ability to support weight of head. *P 5 0.05.

mean (SEM). Pilot studies confirmed that there was no difference in BDNF and NT3 mRNA expression in un- operated naive rats as compared to sham-injured rats that were surgically prepared for cortical impact injury.

RESULTS A Moderate Level of TBI Was Produced by Lateral Cortical Impact Injury

Assessments of transient behavioral changes con- firmed that acute neurological deficits were produced by cortical impact injury. The duration of each index of behavioral suppression in injured rats (n = 15) was sig- nificantly prolonged, as compared to sham-injured rats (n = 3, Fig. 1). These findings corroborate previous studies, which report similar behavioral suppression fol- lowing cortical impact injury in rats (Dixon et al., 1991; Yang et al., 1993).

160 Yang et al.

In Situ Hybridization Studies in Sham Animals Indicated Hippocampal BDNF and NT3 mRNA Were Moderately Expressed

In sham control rats, hybridization signals corre- sponding to antisense BDNF mRNA were detected in both the hippocampus and neocortex, while antisense NT3 probe binding was evident in the hippocampus but not in the neocortex (Fig. 2). Hippocampal BDNF mRNA was primarily distributed in the dentate gyrus and CAI-CA4 fields. NT3 mRNA expressed relatively higher levels in the dentate g y m , CA2, and medial CA1 regions (Fig. 2). Sense probes for BDNF and NT3 did not detect binding (data not shown).

BDNF mRNA But Not NT3 mRNA Expression Was Increased in the Rat Ipsilateral Neocortex and Bilateral Hippocampus After Injury

In situ hybridization studies showed significantly increased expression of BDNF mRNA in the ipsilateral neocortex at 1, 3, and 5 hr after lateral cortical impact injury, although no increases of BDNF mRNA in the contralateral neocortex were detected at these time points (Figs. 2, 3). Induced expression of BDNF mRNA ap- peared diffusely throughout the neocortex, ipsilateral to the site of injury (Fig. 2). Increased expression was es- pecially apparent in the regions adjacent to compressed and contused cortex, a consistent morphopathological characteristic of this model (Dixon et al., 1991; Taft et al., 1993; Posmantur et al., 1994). Increased expression of BDNF mRNA was detected bilaterally in the hippo- campus by in situ hybridization at 1, 3, and 5 hr after lateral cortical impact injury (Figs. 2, 3). Hippocampal expression appeared to be restricted to cell layers and was greatest in the dentate gyrus and CAI-CA4 regions (Fig. 2). Patterns of NT3 mRNA expression in injured rat brains were similar to those in sham-injured rat brains. NT3 mRNA was expressed in the dentate gyrus, CA2, and medial CA1 regions after TBI. However, we did not detect significant changes of the levels of NT3 mRNA in these regions after injury, as compared to sham injury controls (Figs. 2, 3).

DISCUSSION Using in situ hybridization methods, we studied

BDNF and NT3 mRNA expression in a lateral cortical impact injury rodent model (Dixon et al., 1991). To obtain reliable data on co-expression of neurotrophic genes, ad- jacent coronal sections from the same rat brain were hybridized in situ with BDNF and NT3 cRNA probes. Quantitative in situ hybridization analyses detected rapid increases of BDNF mRNA in the ipsilateral neocortex and bilateral hippocampus 1, 3, and 5 hr after TBI. NT3

NT-3 BDNF

Sham

5 m

30 m

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3 h

5 h

Fig. 2 . Expression of BDNF and NT3 mRNA in representative adjacent sections of the rat brain examined by in situ hybrid- ization after lateral cortical impact injury. In the sham control rat, levels of mRNA for BDNF were detected in the bilateral hippocampus and neocortex. In the hippocampus, BDNF mRNA was primarily distributed in dentate gyrus and CAI- CA4 fields. Expression of mRNA for NT3 was evident in the hippocampus but was not apparent in the neocortex. In the hippocampus, NT3 mRNA was located in dentate gyrus, CA2, and medial CAI fields. Arrows indicate sites of injury.

mRNA did not manifest statistically significant changes following TBI. Our results provide evidence that TBI does not cause decreased expression of NT3 mRNA in association with increased expression of BDNF mRNA,

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Time After Injury Fig. 3. Semiquantitative analysis of time-courses of BDNF and NT3 mRNA expression in the neocortex, hippocampal dentate gyrus, and CAl-CA4 after cortical impact injury in rats. Expression of BDNF and NT3 mRNA in the rat brain was examined by in situ hybridization. Levels of BDNF and NT3 mRNA expression were quantified from adjacent sections by a computerized image analysis system. Data for injured rats are expressed as mean percentages of the sham-injured control group, f SEM. Each value represents the average of 3 differ- ent animals. Statistical analysis of changes in density levels

between groups was carried out using one-way analysis of variance (ANOVA). *P 5 0.05. In the injured rat, BDNF and NT3 mRNA levels did not change 5 min and 30 min after injury in the rat brain, whereas BDNF mRNA, but not NT3 mRNA, expression was increased in the ipsilateral neocortex, bilateral hippocampal dentate gyrus, and CAl-CA4 regions. BDNF mRNA expression was increased at 1, 3, and 5 hr after injury. However, NT3 mRNA was not significantly changed at these time-points after lateral cortical impact injury.

162 Yang et al.

as previously reported in animal models of ischemia (Lindvall et al., 1992; Takeda et al., 1992, 1993) and seizures (Ernfors et al., 1991; Isackson et al., 1991; Gall and Lauterborn, 1992; Rocamora et al., 1992).

In contrast to our study employing a rodent model of TBI, previous studies have reported that, along with increased BDNF mRNA, CNS insults often cause de- creased NT3 mRNA expression. NT3 mRNA signifi- cantly decreased following brief periods of ischemia (Lindvall et al., 1992; Takeda et al., 1992, 1993), sei- zures (Ernfors et al., 1990; Isackson et al., 1991; Gall and Lauterborn, 1992; Rocamora et al., 1992), and in- sulin-induced hypoglycemic coma (Lindvall et al., 1992). Why cortical impact injury and other CNS insult paradigms produce varying patterns of gene expressions, including differences in NT3 gene expression, is not known. However, the absence of significant changes in NT3 mRNA expression after TBI does provide evidence that mechanisms regulating neurotrophin expression fol- lowing TBI differ from mechanisms regulating neurotro- phin expression after other CNS insults.

Our findings of increased BDNF mRNA expres- sion in a controlled lateral cortical impact injury model corroborate previous studies of BDNF mRNA expression after CNS injury. A number of studies have shown that BDNF mRNA expression can be induced by experimen- tal CNS insults such as ischemia (Lindvall et al., 1992; Takeda et a]., 1993), seizures (Gall and Isackson, 1989; Isackson et al., 1991; Gall and Lauterborn, 1992; Ro- camora et al., 1992), hypoglycemic coma (Lindvall et al., 1992), glutamate-mediated spreading depression (Kokaia et al., 1993), and glucocorticoid-associated stress (Smith et al., 1995). However, limitations in the accuracy of in situ hybridization technique comparisons of relative increases in BDNF mRNA observed in dif- ferent studies. In general, the regional and temporal characteristics of BDNF expression reported by us are similar to changes in BDNF seen in other injury models. Although the cellular and molecular mechanisms in- volved in regulating the expression of these genes are not well understood, changes in neuronal activity have been reliably associated with neurotrophin gene expression. For example, BDNF mRNA expression is inducible by depolarization (Ghosh et al., 1994; Lu et al., 1991; Zafra et al., 1990), can be enhanced by stimulating the gluta- mate receptors (Gall and Isackson, 1989; Zaal et al., 1993), and can be produced by increased intracellular calcium levels (Lindvall et al., 1992; Ghosh et al., 1994). Accumulating data from experimental studies of TBI (reviewed in Hayes et al., 1992; McIntosh, 1993) indicate that TBI produces massive depolarization (Katayama et al., 1990), increased release of glutamate (Faden et al., 1989; Katayama et al., 1990), and calcium accumulation in the rat brain (Fineman et al., 1993;

Smith et al., 1993). Our results are consistent with the hypothesis that neuronal excitation (Gall and Isackson, 1989; Gall et al., 1991a,b; Springer et al., 1994) and increased intracellular levels of calcium coupled to glu- tamate receptor stimulation (Bessho et al., 1993; Kokaia et al., 1993; Springer et al., 1994) could contribute to increases in BDNF mRNA after CNS insults.

Our in situ hybridization studies detected signifi- cant bilateral increases in BDNF mRNA in cells in the regions of the dentate gyrus and CAl-CA4 of the hip- pocampus. The highest levels of BDNF mRNA were consistently seen in the dentate region. BDNF mRNA levels increased only ipsilateral to the injury site in the neocortex after controlled lateral cortical impact injury. This finding is consistent with previous observations that similar regions of the hippocampus are more vulnerable to TBI than the neocortex (Jenkins et al., 1989; Lyeth et al., 1990; Hayes et al., 1992; Lowenstein et al., 1992; Taft et al., 1992; Yang et al., 1993, 1994). The patho- physiological significance of substantial increases of BDNF mRNA in the hippocampal dentate gyrus is un- known. Whether the vulnerability of the dentate gyrus to injury is associated with a compensatory neurotrophic response requires further investigation. Although precise mechanisms mediating hippocampal responses to me- chanical injury are not known, bilateral changes in the hippocampus following unilateral cortical impact injury may be a characteristic of cortical impact injury. We have consistently observed a contralateral hippocampal response to ipsilateral injury in this lateral cortical impact injury rat model (Yang et al., 1994).

It is possible that BDNF mRNA expression is as- sociated with expression of mRNA for the immediate early gene, c-fos (Kokaia et al., 1993). TBI produces a similar regional profile of c-fos mRNA expression in the rat brain (Yang et al., 1994). We recently reported that c-fos mRNA expression following TBI occurs as early as 5 min after injury in the ipsilateral cortex and precedes NGF mRNA expression (Yang et al., 1995). Thus, ex- pression of c-fos mRNA also precedes expression of BDNF mRNA after TBI and occurs in similar brain re- gions. BDNF mRNA and c-fos mRNA coexpression is similar in other CNS insult paradigms. Spreading depres- sion also leads to c-fos mRNA and BDNF mRNA ex- pression in the same brain region (Kokaia et al., 1993). Unilateral needle insertion or saline injection into the dorsal hippocampus can cause increased BDNF mRNA in granular neurons of the dentate gyrus, which is pre- ceded by an increase in c-fos mRNA in the same brain regions (Ballarin et al., 1991). However, there is cur- rently no direct evidence that BDNF mRNA expression can be induced by c-fos gene expression.

The significance of BDNF mRNA expression after TBI is unknown. However, BDNF has been shown to

Increased BDNF, Not NT3, mRNA in Injured Rat Brain 163

increase the survival in cultures of retinal ganglion cells, basal forebrain cholinergic neurons, and ventral mesen- cephalic dopaminergic neurons (Barde et al., 1980; Anderson et al., 1990; Lyeth et al., 1990; Hyman et al., 1991; Knusel et al., 1991). Recent studies also suggest that BDNF is required for activity-dependent survival of cortical neurons (Ghosh et al., 1994). Therefore, it is possible that increased BDNF gene expression may be a neuroprotective response to injury, although the func- tional implications of increases of BDNF mRNA levels following some brain insults remain to be fully eluci- dated.

In conclusion, this paper provides evidence that cortical impact injury produces rapid increases in BDNF mRNA expression in the rat brain without changes in NT3 mRNA expression, a finding that differs from pre- vious reports on ischemia and seizures. Our results also suggest that increased BDNF may be an important me- diator of the neuronal pathophysiological process after TBI, while NT3 may be irrelevant to these early re- sponses.

ACKNOWLEDGMENTS This work was supported by grants PO1 NS 31998

and ROl NS 21458 from the National Institutes of Health.

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