Subventricular Zone Neural Precursor Cell Responses after Traumatic Brain Injury and Binge Alcohol in Male Rats

Abstract

Traumatic brain injury (TBI) is a major cause of disability worldwide. Additionally, many TBI patients are intoxicated with alcohol at the time of injury, but the impact of acute intoxication on recovery from brain injury is not well understood. We have previously found that binge alcohol prior to TBI impairs spontaneous functional sensorimotor recovery. However, whether alcohol administration in this setting affects reactive neurogenesis after TBI is not known. This study therefore sought to determine the short and long term effects of pre-TBI binge alcohol on neural precursor cell responses in the subventricular zone (SVZ) following brain injury in male rats. We found that TBI alone significantly increased proliferation in the SVZ as early as 24 hours after injury. Surprisingly, binge alcohol alone also significantly increased proliferation in the SVZ after 24 hours. However, a combined binge alcohol and TBI regimen resulted in decreased TBI-induced proliferation in the SVZ at 24 hours and 1 week post-TBI. Furthermore, at 6 weeks after TBI, binge alcohol administered at the time of TBI significantly decreased the TBI-induced neuroblast response in the SVZ and the rostral migratory stream (RMS). The results from this study suggest that pre-TBI binge alcohol negatively impacts reparative processes in the brain by decreasing short-term neural precursor cell proliferative responses as well as long-term neuroblasts in the SVZ and RMS.

INTRODUCTION

The incidence of traumatic brain injury (TBI) in our society is striking with 2.8 million occurrences annually (Taylor 2017). Many survivors are faced with debilitating outcomes such as deficits in sensory or motor function and increased risk for mood and dementia disorders (CDC 2017; Langlois et al. 2006). Additionally, up to 53% of TBI patients presenting to the emergency department have blood alcohol levels above the legal limit (Beaulieu-Bonneau et al. 2017; Talving et al. 2010). Binge drinking has become one of the most common modes of alcohol abuse according to a 2017 CDC report (CDC 2017). The National Institute on Alcohol Abuse and Alcoholism (NIAAA) defines binge drinking as a mode of consumption that results in a blood alcohol level of greater than 80mg/dl, which is typically achieved by having 5 drinks for men and 4 drinks for women in two hours (Olthuis et al. 2011). Our group has shown that in a rat model of TBI and binge alcohol, recovery in a skilled sensorimotor task was slower and the recovery plateau was lower in alcohol-exposed animals (Vaagenes et al. 2015) as also reported in clinical studies (Corrigan 1995; Gurney et al. 1992; Joseph et al. 2015; Schutte and Hanks 2010) although others have found a benefit (Chandrasekar et al. 2018; Chandrasekar et al. 2017; Lundgaard et al. 2018).

The cellular and molecular underpinnings of this binge pattern of alcohol consumption on TBI recovery has yet to be elucidated, and a more fundamental understanding of TBI and binge alcohol will help in designing better therapeutic interventions for patients. Accordingly, it is thought that one way in which the brain compensates after an injury is to increase neurogenesis in various neurogenic regions such as the subventricular zone (SVZ) (Alagappan et al. 2009; Chirumamilla et al. 2002; Dash et al. 2001; Sun et al. 2005). In rodents, immature neurons from the adult SVZ are known to migrate through the RMS to continually supply the olfactory bulb (OB) with new neurons (Lim and Alvarez-Buylla 2016). Following injury, neurogenesis may improve functional recovery by incorporation of new neurons (Shiromoto et al. 2017; Zhao et al. 2008), although the role of adult neurogenesis across species (Faykoo-Martinez et al. 2017) and particularly in humans (Sorrells et al. 2018) is under debate. The goal of this study was to determine how TBI affects neurogenesis in the rat SVZ and if an alcohol binge prior to TBI leads to a reduction in neurogenesis. While the SVZ is not the only area of adult neurogenesis, its location close to the sensorimotor cortex being damaged by the TBI as done in this study increases the likelihood that it will respond to the injury (Ramaswamy et al. 2005; Richardson et al. 2007). Our results show that the combination of binge alcohol and TBI significantly reduced the early proliferative response within the SVZ and decreased the neuroblast response in the SVZ and RMS.

Methods

Ethics statement

Animal use was approved by the Institutional Animal Care and Use Committee (IACUC) of Edward Hines Jr. Veterans Affairs Hospital permit #H13-001.

Animal subjects

2 month-old adult male Sprague Dawley rats (Harlan, Indianapolis, IN) were used in this study (76 total animals) (Table 1). Male rats were used in this study because TBI during alcohol intoxication is more commonly seen in male humans (CDC 2017). Rats were housed two in each cage in a fully accredited animal care facility with a 12-hour light/dark cycle. Food and water was available ad libitum. All animals were number coded by a person not participating in the experiment, who also performed randomization. Investigators conducting surgeries, lesion analysis and stereology were blinded to the animal treatment groups throughout the experiments. Codes were revealed after all the data points were analyzed. See Fig. 1A for the experimental design.

Table 1. Experimental Groups.

Animal number (n) and groups for the three different time points examined (24 hours, 1 week and 6 weeks respectively). Animals were randomly assigned to each of the 4 experimental groups (1-4), either with alcohol or vehicle and TBI or sham surgery.

Group	Alcohol	TBI	24 hours	1 week	6 weeks
1	−	−	6	6	6
2	−	+	6	7	7
3	+	−	6	6	6
4	+	+	6	7	7
Subtotal			24	26	26
Total			76

Figure 1:

A. Timeline for the experimental design. B. Representative image of a brain that sustained a TBI and coronal sections (B’) of a TBI in the sensorimotor cortex. Arrows point to the location of the TBI lesion. C. Box and whisker plot of lesion size at various time points post-TBI. The horizontal line within the box indicates the median, boundaries of the box indicate the 25^th and 75^th percentile, and the whiskers indicate the highest and lowest values of the results. There was no statistical difference between groups at any time point, Student’s t-test.

Alcohol administration

Rats were administered one dose of alcohol per day for three consecutive days at 9 AM by gastric gavage (3g/kg/dose/day, i.g. 40% ABV). We used this dose to achieve a blood alcohol concentration (BAC) of greater than 80mg/dL as is seen in many patients who sustain a TBI (Beaulieu-Bonneau et al. 2017). A curved stainless steel gavage needle with a smooth rounded ball head attached to a 5cc syringe was used to administer the alcohol. All animals tolerated the gavage procedure well with no adverse effects. Control animals received an equal volume of water using an identical dosing schedule.

TBI by controlled cortical impact (CCI)

CCI was performed as previously described (Vaagenes et al. 2015). Briefly, one hour after the last gavage administration, rats were anesthetized using 75mg/ml of isoflurane delivered as an inhalant (5% isoflurane in 100% oxygen). Animals were secured in a stereotaxic frame, a midline incision was made in the scalp and the skin was retracted. Using a trephine, a 5mm diameter disc of skull was removed above the forelimb area of the sensorimotor cortex (trephine was centered at the coordinate 1mm anterior, 1.5mm lateral from bregma) (Neafsey et al. 1986) of the right hemisphere and the dura was carefully removed. The TBI was delivered using a double acting magnetic piston mounted on a stereotaxic crossbar, angled for cortical impact (diameter: 3 millimeters, velocity: 2.5 meters/second, depth: 2 millimeters, dwell time: 250 milliseconds) (Impact One, MyNeurolab, St. Louis, MO). Sham animals were treated identically (anesthetized, skin incision and trephination), apart from the piston. After CCI, 5 rats were excluded due to their lesion sizes being more than 2 standard deviations from the group mean. In our model, this injury leads to a prolonged deficit in the skilled forelimb reaching task in the forelimb contralateral to the impaction (Vaagenes et al. 2015).

Blood alcohol level quantification

100μL of blood was drawn from the tail vein of each rat immediately following the TBI or sham surgery. Blood was then allowed to clot on ice for five minutes and centrifuged to separate the serum. Alcohol concentration was measured using an alcohol reagent set (Pointe Scientific, Canton MI, USA) according to manufacturer’s instructions. The serum samples were de-proteinized using 6.25% trichloroacetic acid; 0.9% sodium chloride was used as the non-serum blank. Samples were loaded onto 96 well plates at a sample/reagent ratio of 1:201, incubated at 30°C for 5 minutes, and the absorbance (340nm) was read at 30°C. A standard curve was generated for each assay run from the known ethanol samples. Sample measurements were done in duplicate and the values averaged for each animal. Known high and low alcohol controls were always run along with unknown samples and the resultant alcohol concentration values for the controls were always within acceptable range (+/− 5%).

Bromodeoxyuridine (BrdU) administration

We utilized two separate BrdU injection paradigms (Fig. 1A). BrdU was prepared at 20mg/mL in sterile saline. To measure short-term cellular proliferation, a single injection of BrdU at lOOmg/kg (i.p.) was injected immediately after TBI followed by euthanasia 24 hours later. A single injection of BrdU labels cells in S-phase for approximately two hours after injection (Taupin 2007). For the longer time-course groups, which allow for the determination of neural precursor cell response and the phenotype of newborn cells, BrdU was injected at lOOmg/kg (i.p.) starting immediately following TBI and continuing once a day for the next six days. Rats were briefly anesthetized with isoflurane during BrdU injections to minimize discomfort.

Perfusion, tissue processing, and histology

At either 24 hours, 1 week or 6 weeks post-TBI, rats were overdosed with Euthasol (phenytoin/pentobarbital; 390 mg/kg i.p.) and transcardially perfused with cold heparinized saline followed by 4% paraformaldehyde (PFA). Brains were extracted and post-fixed overnight at 4°C in 4% PFA, then cryoprotected in 30% sucrose in phosphate buffer pH 7.4 until sinking. Brains were frozen, coronally cryosectioned at 40μm thickness and sections stored in ethylene glycol at −20°C until use. We performed antigen retrieval (Tang et al. 2007) as follows: tissue sections were subjected to high heat (99-100°C) in a 10 mM sodium citrate (pH 6.0) solution for 15 minutes . The following primary antibodies were used: mouse IgG2a anti-BrdU (Pierce; 1:4000, RRID: AB_10986341) and rabbit anti-doublecortin (DCX) (Cell Signaling Technology; 1:1000, RRID: AB_10693771). Primary antibodies were diluted in phosphate- buffered saline (PBS) pH 7.4 plus 0.2% Tween-20. For immunostaining, tissue sections were then incubated in primary antibody solution overnight at 4° C with gentle agitation. The following day, the tissue sections were extensively washed in PBS/0.2% Tween-20. The sections were then incubated in secondary antibodies (AP-Goat Anti-Rabbit, Invitrogen, RRID: AB_228339; Biotin Conjugated Goat Anti-Mouse IgG2a Specific, Jackson Laboratory, RRID: AB_2338572; Alexa Fluor 488 Goat Anti-Mouse IgG2a, RRID: AB_141618; 568 Goat Anti-Rabbit, Life Technology, RRID: AB_143011; all 1:500) in the same dilution buffers listed above for 2 hours at room temperature with shaking, and then washed. For fluorescent imaging, nuclei were counterstained with DAPI, then mounted on gelatin-subbed slides and coverslipped with Fluoromount G anti-fade mounting media. For signal detection with the chromogenic substrate diaminobenzidine (DAB), tissue sections were incubated in avidin-biotin- peroxidase complex (Vector Laboratories) for 1 hour at room temperature per manufacturer’s instructions. Lastly, sections were reacted in nickel-enhanced DAB in the presence of hydrogen peroxide to visualize the target antigens.

Stereology

Unbiased stereology is a method of estimating properties (number, length, volume, etc.) of three-dimensional structures from tissue sections without making assumptions about the size, shape, or orientation of the objects of interest (Napper 2018; Peterson 1999). As previously published (Shepherd et al. 2017), we performed stereology using a software-based optical fractionator probe. Briefly, sections encompassing the RMS and anterior SVZ (between 4.00 mm anterior and 0.80 mm posterior to bregma, (Paxinos and Watson 1998)) were examined (4 sections taken from the same anatomical level/subject). Outline tracings of the SVZ were performed under a 2.5X objective. Both the sampling grid size (64×150μm) and the dissector window size (25×25μm) were optimized to provide adequate sampling of the entire SVZ. Under high magnification of 40X/0.75 NA objective, the optical dissector window was systematically overlaid over the SVZ area and cells within the dissector window were counted. Single-labeled BrdU⁺ and double-labeled BrdU⁺/Dcx⁺ cells were counted and the accuracy of double labeling was validated using confocal microscopy (Fig 3). Single-labeled BrdU⁺ cells showed typical nuclear staining (Fig 3A’”-A””, white arrows) while double-labeled BrdU⁺/Dcx⁺ cells showed Dcx staining surrounding the BrdU labeled nuclei (Fig 3A’”-A””, yellow arrows). To avoid oversampling, we excluded the uppermost and lowermost focal planes as is typically done. All stereological cell counting was performed on a MBF Bioscience Leica DM400B microscope with Stereoinvestigator software version 9.0.

Figure 3:

Binge alcohol significantly decreased neuronal differentiation in the ipsilesional SVZ and RMS at 6 weeks after injury. Representative immunofluorescence staining demonstrates labeling of BrdU⁺ (green) and DCX⁺(red) cells in the ipsilesional SVZ. The arrow in A points to a yellow box which is the area magnified in A’ and A”. A’ and A”, scale bar = 50 μm. A’“ and A”“, scale bar = 10μm. Yellow arrows point to double-labeled cells (new immature neurons) as indicated in the orthogonal views. White arrows point to single-labeled BrdU⁺ cells. (B-G) Box and whisker plots of stereology quantification of BrdU⁺ cells in the ipsi and contra-lesional SVZ and RMS. The horizontal line within the box indicates the median, boundaries of the box indicate the 25th and 75th percentile, and the whiskers indicate the highest and lowest values of the results. B. Binge alcohol did not affect the SVZ BrdU⁺/DCX-cell number. D. Binge alcohol significantly decreased the SVZ BrdU⁺/DCX⁺ cell number in animals with TBI. (C,E). Percent of BrdU⁺/DCX⁻ and BrdU⁺/DCX⁺ relative to total BrdU⁺ labeled cells in the SVZ respectively, error bars=SD. F. Binge alcohol did not affect the BrdU⁺/DCX⁻ RMS cell number. G. Binge alcohol significantly decreased the RMS BrdU⁺/DCX⁺ cell number in animals with TBI. Two-way ANOVA, post-hoc within-group analysis,* denotes p≤0.05, ** p≤0.01,and *** p≤0.001.

Lesion analysis

Alternating sections were mounted on gelatin-coated slides and stained for Nissl substance (Fig. 1B). Nissl stained slides were then scanned at high resolution using a flatbed scanner and imported into Adobe Photoshop CS5. The number of pixels in the intact and lesioned hemispheres was measured from sections between 1.4mm anterior and 2.5mm posterior to bregma, which encompassed the extent of the lesion. The lesion size was quantified as a percentage of the unlesioned contralateral hemisphere (the total area of the uninjured contralateral hemisphere minus total area of the injured (ipsilesional) hemisphere, divided by the intact hemisphere) (Papadopoulos et al. 2002) (Fig. 1C).

Statistical analysis

All data analysis was performed using either Minitab version 17 (Minitab, Inc. State College, Pennsylvania, USA) or Graphpad Prism version 5.0 (GraphPad Software, San Diego California, USA). Lesion volume comparisons were done using Student’s t-test with α=0.05 as the cutoff for statistical significance. BrdU stereological counting results for the 24 hours post-TBI time point were analyzed by using a general linear model two-way ANOVA (F tests) to detect main effects and interactions between alcohol treatment and TBI injury; residual analysis revealed that raw data were not in need of any transformation. For 1 week post-TBI groups, cell count data was transformed on the natural log scale. For 6 weeks post-TBI groups, the cell count data was transformed using the corresponding optimal (Box-Cox) transformation (Altman 1990). All data transformations were necessary to satisfy the Normality-and-Constant-Variance requirements (Box and Cox 1964). At all time points, to compare if group means were significantly different, WITH-IN group analysis was performed using regression analysis. The actual P values were reported unless the number was less than 0.005, in which case P<0.005 was indicated.

Results

Alcohol gavage administration achieved a high blood alcohol level (BAL)

The mean BAL at the time of TBI was 156.1mg/dL +/− 8.3mg/dL. This level is consistent with previous reports using the same animal strain, (Livy et al. 2003; Vaagenes et al. 2015) and is approximately twice the legal intoxication limit in humans (Olthuis et al. 2011).

Binge alcohol administration prior to TBI did not affect lesion size

The lesion was confined to the right sensorimotor cortex, with the center of the lesion at approximately 1.5 mm anterior and 2.5 mm lateral from bregma (Fig. 1B,B’). There was no significant difference in lesion size between vehicle and alcohol treated groups at any time point post-TBI (Fig. 1C).

Binge alcohol combined with TBI decreased proliferation in the SVZ at 24 hours

It has been reported that there is a general increase in SVZ proliferation post-TBI, (see Chang et al. 2016 for review). We observed that the SVZ responded robustly through upregulation of the number of proliferating cells to TBI starting as early as only 24 hours after injury (Fig. 2A,C). What was unknown, however, was how binge alcohol administration prior to TBI affected the SVZ proliferative response. There was a statistically significant Injury (TBI) × Treatment (alcohol) interaction for ipsilesional F;u₃)=10.14; p<0.005 and contralesional F(_1,23)=13.77; p<0.005 BrdU⁺ cell counts. Pre-planned multiple comparisons showed that the mean number of proliferating cells in the SVZ of the Vehicle/TBI group was higher than that of the Vehicle/Sham group for both ipsilesional (F(_1,23)=10.94; p<0.005) and contralesional (F(_1,23)=9.44; p=0.006) hemispheres (Fig. 2E), indicating that TBI alone induced an approximately 4 fold increase in cell proliferation in the SVZ. The mean number of proliferating cells in the Alcohol/TBI group was lower than that of the Vehicle/TBI group in ipsilesional (F(_1,23)=4.85; p=0.039) and contralesional (F(_1,23)=6.79; p=0.016) hemispheres, i.e, when alcohol was administered prior to the TBI, proliferation was reduced bilaterally in the SVZ by about 2 fold (Fig. 2B,D, E). Interestingly, the number of proliferating cells of the Alcohol/Sham group was 3 fold higher than that of the Vehicle/Sham group in ipsilesional (F(_1,23)=4.88; p=0.038) and contralesional (F(_1,23)=7.47; p=0.012) hemispheres, (Fig. 2E), indicating that alcohol alone stimulated proliferation in the SVZ bilaterally.

Figure 2:

(A-D) Representative images of the ipsilesional SVZ at 24 hours after TBI. Binge alcohol significantly increased SVZ proliferation in sham groups (A,B) and decreased proliferation in TBI groups (C,D). LV=lateral ventricle, CC=corpus callosum, ST= striatum, SVZ=subventricular zone. Dashed line in insert (B’) encloses BrdU⁺ nuclei of SVZ cells. Scale bar=50μm. (E-G) Box and whisker plots of stereology quantification of BrdU⁺ cells in the ipsi and contra-lesional SVZ at various time points post-TBI. The horizontal line within the box indicates the median, boundaries of the box indicate the 25^th and 75^th percentile, and the whiskers indicate the highest and lowest values of the results. E. At 24 hours after injury, binge alcohol significantly increased SVZ proliferation in sham groups and decreased proliferation in TBI groups bilaterally. F. At one week after injury, binge alcohol significantly decreased proliferation in the TBI groups bilaterally. G. At 6 weeks after injury, binge alcohol had no effect on SVZ proliferation. However, TBI still had a proliferative effect on the ipsilesional SVZ. Two-way ANOVA, post-hoc within-group analysis, * denotes p≤0.05, ** denotes p≤0.01, *** denotes p≤0.001.

Binge alcohol combined with TBI decreased proliferation in the SVZ at 1 week

Various groups have reported peak proliferation in the SVZ to occur at one week after TBI (Bye et al. 2011; Gotts and Chesselet 2005; Szele and Chesselet 1996). Our question was whether binge alcohol prior to TBI still affected SVZ proliferation at this time point. We did not detect a statistically significant main effect of either Injury (TBI) or Treatment (Alcohol) on proliferation. Furthermore, there was no significant Injury × Treatment interaction for either ipsilesional or contralesional BrdU⁺ cell counts. However, multiple comparisons (WITH-IN analysis) showed that the mean BrdU⁺ cell count in the Alcohol/TBI group was significantly lower than that of the Vehicle/TBI group in ipsilesional (F(_1,25)=5.85; p=0.024) and contralesional (F(_1,25)=5.54; p=0.028) hemispheres (Fig. 2F), indicating alcohol combined with TBI significantly reduced proliferation up to 2 fold in the SVZ bilaterally.

Total BrdU⁺ cell count was elevated only in the ipsilesional SVZ post TBI at 6 weeks

We found that there was a statistically significant main effect of Injury (TBI), F_(1,25)=137.54; p<0.005 on the BrdU⁺ cell count of the ipsilesional SVZ at 6 weeks post TBI. Multiple comparisons showed that the mean BrdU⁺ cell count of the Vehicle/TBI group was significantly higher than that of the Vehicle/Sham group (F_(1,25)=67.34; p<0.005), (Fig. 2G), and similarly for the Alcohol TBI group vs. Alcohol Sham group (F_(1,25)=70.22; p<0.005), indicating that TBI alone induced more than a 4 fold increase in the number of proliferating cells in the ipsilesional SVZ. However, alcohol had no effect on proliferation at the 6 week time point. Furthermore, in contrast to the ipsilesional SVZ, no statistically significant effects of Injury, Treatment or Injury x Treatment interaction in the contralesional SVZ were detected (Fig. 2G).

Binge alcohol did not affect the number of BrdU⁺/DCX⁻ cells in the SVZ and RMS at 6 weeks post-TBI

In the adult, SVZ precursor cells proliferate and can differentiate into mature neurons (Kempermann 2011). These SVZ derived neurons express developmental stage specific markers that are also seen during embryonic development. Doublecortin (DCX) is a microtubule-associated protein specifically expressed by newborn migratory neurons (Couillard-Despres et al. 2005; Kempermann 2011), and is thus a reliable marker for neuronal differentiation (Francis et al. 1999). At 6 weeks post TBI, two distinct populations of cells in the SVZ and RMS were observed: those that were BrdU⁺DCX⁻ and those that were BrdU⁺/DCX⁺(Fig. 3A-A”” & Supp. Fig. 1). BrdU⁺/DCX⁻ cells are early-proliferated cells that either remained undifferentiated or differentiated into the non-neuronal lineages. We detected that there was a statistically significant main effect of Injury (TBI), F(_1,25)=83.19; p<0.005, on the number of BrdU⁺/DCX⁻ cells only in the ipsilesional SVZ. Multiple comparisons showed that the means of BrdU⁺/DCX⁻ cell number of the Vehicle Sham and Alcohol Sham groups were lower than that of the Vehicle TBI and Alcohol TBI groups, (F(_1,25)=38.59; p<0.005) and (F(_1,25)=44.72; p<0.005) respectively, (Fig. 3B). This suggests that TBI stimulated the proliferation and differentiation of cells in the non-neuronal lineage such as astrocytes. However, it is also possible that BrdU⁺/DCX⁻ cells could be microglia or infiltrating blood-borne macrophages as these types of immune cells have been found to proliferate after TBI (Lalancette-Hébert et al. 2007).

The RMS is the anterior extension of the SVZ which is a path that migratory immature neurons take toward the OB. Here, we did not detect a statistically significant main effect of either Injury (TBI) or Treatment (Alcohol) on the number of BrdU⁺/DCX⁻ cells. Furthermore, there was no significant Injury × Treatment interaction for either ipsilesional or contralesional BrdU⁺/DCX⁻ cell counts.

Binge alcohol decreased BrdU⁺/DCX⁺cells in the SVZ and RMS at 6 weeks post-TBI

BrdU⁺/DCX⁺ double-labeled cells are immature migratory neurons, and these cells typically migrate toward the olfactory bulbs (Lim and Alvarez-Buylla 2016). Alcohol exposure during fetal development has been shown to decrease this population of migratory neurons in rodents (Camarillo and Miranda 2007; Miller 1986; Zhou et al. 2001). We found that TBI alone stimulated an increase in the number of immature migratory neurons in the SVZ at 6 weeks after injury. We detected a statistically significant main effect of Injury (TBI) on BrdU⁺/DCX⁺ cell numbers in the SVZ bilaterally, F(_1,25)=117.12; p<0.005 for ipsilesional and F(_1,25)=18.50; p<0.005 for contralesional. Additionally, there were significant Injury × Treatment interactions; F(_1,25)=5.43; p=0.029 for ipsilesional and F(_1,25)=6.16; p=0.021 for contralesional. Multiple comparisons showed that the mean BrdU⁺/DCX⁺ cell number in the SVZ of the Vehicle/Sham group was much lower than that of the Vehicle/TBI group, for ipsilesional (F(_1,25)=86.49; p<0.005 ) and contralesional (F(_1,25)=23; p<0.005 ) hemispheres, and likewise for the Alcohol/Sham versus Alcohol/TBI comparison in the ipsilesional hemisphere (F(_1,25)=36.06; p<0.005 ), (Fig. 3D), indicating that TBI alone stimulated a large increase in neuroblasts bilaterally by up to 11 fold. Furthermore, TBI decreased the relative percentage of BrdU⁺/DCX⁻ cells and shifted it toward an increased percentage of BrdU⁺/DCX⁺ cells (Fig. 3C & 3E). Among the TBI injured rats, the binge alcohol-exposed (Alcohol/TBI group) had a significantly lower mean number of BrdU⁺/DCX⁺ cells than the control (Vehicle/TBI group) in the ipsilesional SVZ, (F_1,25)=9.17; p=0.006), (Fig. 3A’, 3A”, 3D), indicating that alcohol combined with TBI decreased neuronal differentiation in the SVZ at 6 weeks after injury.

In the RMS, there was a statistically significant main effect of Injury (TBI) on BrdU⁺/DCX⁺ cell numbers bilaterally, F(_1,25)=30.67; p<0.005 for ipsilesional and F(_1,25)=7.538; p=0.0125 for contralesional; and a significant main effect of Treatment (Alcohol) bilaterally F(_1,25)=18.37; p<0.005 for ipsilesional and F(_1,25)=4.721; p=0.0420 for contralesional hemispheres. Additionally, there were significant Injury × Treatment interactions; F(_1,25)=22.95; p<0.005 for ipsilesional and F(_1,25)=8.713; p=0.0079 for contralesional hemispheres. Multiple comparisons showed that the mean BrdU⁺/DCX⁺ cell number in the RMS of the Vehicle/Sham group was much lower than that of the Vehicle/TBI group, F(_1,25)=74.44; p<0.005 for ipsilesional and F(_1,25)=9.803; p=0.0166 for contralesional hemispheres, (Fig. 3G), indicating that TBI alone stimulated a large neuroblast response bilaterally. Among the TBI injured rats, the binge alcohol-exposed (Alcohol/TBI group) had a significantly lower mean number of BrdU⁺/DCX⁺ cells than the control (Vehicle/TBI group) bilaterally in the RMS, F(_1,25)=68.99; p<0.005 for ipsilesional and F(_1,25)=9.517; p=0.0177 for contralesional hemispheres, (Fig. 3G & Supp. Fig. 1), indicating that alcohol combined with TBI decreased the number of migratory neuroblasts in the RMS at 6 weeks after injury.

DISCUSSION

Our results show that alcohol given for three consecutive days (a binge pattern of alcohol consumption) before a TBI to the forelimb motor cortex results in a significant reduction in SVZ proliferation when measured at 24 hours and 7 days post injury compared to TBI alone. Although binge alcohol did not affect the number of proliferated cells in the SVZ when measured at 6 weeks post injury, it did significantly reduce the number of neuroblasts in the SVZ and RMS. An interesting effect of binge alcohol alone was its effect on increasing SVZ proliferation when measured 24 hours after the last binge episode.

TBI alone increased SVZ proliferation

We observed a general increase in SVZ proliferation post-TBI, and confirmed earlier reports that TBI produced a significant increase in the number of proliferating cells in the SVZ bilaterally when measured at 24 hours and 7 days post injury and ipsilaterally at the 6 week time point, as in agreement with past literature regarding proliferation after TBI in rats (Chang et al. 2016). However, there were some differences in the time-course of this proliferative response in our model as compared to other studies. We found that TBI alone induced up to a 4 fold increase in SVZ proliferation at the 24 hour time point whereas others have reported no change in SVZ proliferation during the first five days after injury. The difference in this result might be due to the type of TBI model used, for instance, one group used the stab wound model to inflict TBI (Tzeng and Wu 1999). Another group used a thermal coagulation model to produce a cortical lesion that was distal to the SVZ and reported that within the first 5 days after injury, there was no change in proliferation (Gotts and Chesselet 2005). In contrast, the model used in the present report, i.e., CCI, produced a focal TBI lesion that was closer to the SVZ, and therefore might explain the marked and almost immediate elevation in SVZ proliferation.

Furthermore, we found that there was no significant difference in SVZ proliferation in TBI versus the control group at one week post-injury indicating that the proliferative maximum had been reached between days 1 and 7, and that the rate of new cell incorporation into the SVZ equaled the rate of migration of cells away from the SVZ. This is similar to reports which used aspiration lesion (Szele and Chesselet 1996) and diffuse TBI models (Bye et al. 2011), where SVZ proliferation reached a maximum of around 2 fold above baseline at 5-7 days post-TBI.

Binge alcohol alone increased short-term SVZ proliferation

We found that binge alcohol alone elicited a bilateral proliferative response in the SVZ, as alcohol only treated animals exhibited up to a 4 fold increase in BrdU⁺ cells at 24 hours compared to controls, similar to the magnitude observed after TBI alone. This is in contrast with previous studies using different alcohol intoxication models in rodents which reported that alcohol caused significant depression in SVZ and subgranular zone (SGZ) proliferation as examined at various time points (5 hours up to 41 days) post alcohol exposure (Anderson et al. 2012; Liu and Crews 2017; Nixon and Crews 2002). However, one report found that discontinuing chronic alcohol administration led to an increase in SGZ cell number at day 2 and 7 after cessation (Anderson et al. 2012). Another study found similar proliferation bursts at 3 days after abstinence in the SVZ using chronic doses of alcohol for extended periods of time (up to 7 weeks), (Hansson et al. 2010). Furthermore, one study found that after discontinuing binge alcohol, there was an increase in the number of BrdU⁺ cells that also expressed Iba-1, a marker for microglia at two days post withdrawal (Nixon et al. 2008). Another group found an increase in BrdU⁺ cells co-labeled with astrocyte markers throughout the cortex post binge alcohol exposure (Helfer et al. 2009). Therefore, it is possible that the increase in the number of proliferative cells (BrdU⁺) that we observed in rats treated with binge alcohol alone was a result of astrocytosis or microgliosis.

Binge alcohol combined with TBI decreased TBI-induced SVZ proliferation

We found that animals receiving binge alcohol had a 3 fold decrease in proliferating cells in the SVZ at 24 hours post-TBI. To our knowledge, we are the first group to show that alcohol combined with TBI resulted in decreased TBI-induced SVZ proliferation. When binge alcohol or TBI were given alone, the result was increased proliferation, but the combination of the two factors led to decreased proliferation in the SVZ. There are several mechanisms that might explain this result, one of which is the neuroinflammatory process. Inflammation is an important component of the secondary injury phase of TBI, (see Woodcock and Morganti-Kossmann 2013 for review) and is thought to be both beneficial and detrimental to CNS recovery (Corps et al. 2015). Furthermore, alcohol by itself is a potent modulator of CNS inflammation. For instance, microglial activation had been observed in rat models of adolescent (McClain et al. 2011) and adult (Marshall et al. 2013) binge alcohol exposure. It is therefore possible that the combined magnitude of inflammation in TBI/binge alcohol animals is higher than in TBI or binge alcohol animals alone. Inflammation can stimulate neurogenesis (Butovsky et al. 2006), however, excessive inflammation may cause the SVZ microenvironment to be non-supportive for neurogenesis (see Ekdahl et al. 2009 for review). For instance, there is strong evidence that pro-inflammatory cytokines such as IL-1b suppress hippocampal neurogenesis (Goshen et al. 2008).

Another possible mechanism in which alcohol might negatively synergize with TBI to attenuate SVZ cell proliferation is via the generation of reactive oxygen species (ROS). During the secondary injury process following TBI, oxidative stress is elevated due to the generation of ROS such as superoxide and peroxinitrite that can damage neurons and glia and disrupt the BBB (Hall et al. 2010). Furthermore, alcohol metabolism in the brain via alcohol dehydrogenase (ADH) and cytochrome P450-2E1 (CYP2E1) has been found to generate free radicals (Haorah et al. 2008) and increase lipid peroxidation (Altura et al. 2002) which are all hallmarks of elevated ROS. Therefore, in TBI and alcohol administered animals, ROS levels could become additive from the two conditions and thereby inhibit cellular proliferation (Le Belle et al. 2011). As previously noted, a low concentration of ROS can stimulate neurogenesis, likely through a mechanism involving elevated NF-kB (Ruiz-Ramos et al. 2009). However, high levels of ROS have been found to inhibit neural stem cells proliferation (Limoli et al. 2006). Furthermore, high levels of ROS could lead to an increased rate of cell death of recently proliferated cells due to an increased rate of DNA damage (Le Belle et al. 2011).

Additionally, neurotrophic factor availability in the CNS could be another mechanism that affects neurogenesis in our model of binge alcohol and TBI. Key neurotrophic factors such as nerve growth factor (NGF), epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and brain derived neurotrophic factor (BDNF) regulate certain critical aspects of neurogenesis (Duman and Monteggia 2006; Kuhn et al. 1997; Zhao et al. 2007). Following TBI, neurotrophic factor activity is increased (Nieto-Sampedro et al. 1982), including astrocyte derived NGF (DeKosky et al. 1994; Goss et al. 1998), and BDNF (Hicks et al. 1997; Rostami et al. 2014; Zhang et al. 2017). Interestingly, various alcohol administration models show disrupted trophic factor activity and function in the CNS (Heaton et al. 1992) including BDNF (Tapia-Arancibia et al. 2001) and NGF receptor (Dohrman et al. 1997), thereby attenuating the trophic factor activity that was increased by TBI.

However, one must be cautious when interpreting BrdU data in terms of proliferation. BrdU is quickly metabolized and is only available for incorporation into proliferating cells for approximately two hours after systemic administration (Nowakowski and Hayes 2000). For this reason, we administered up to 7 daily BrdU injections in order to label proliferating cells during this time period. However, while proliferating cells in the S-phase will incorporate available BrdU, neither a single dose of BrdU (as in the 24 hours survival groups) or multiple doses of BrdU (as in the 7 days and 6 weeks survival groups) will label the total number of proliferating cells. Therefore, the resultant labeled cells should be treated as a relative number, specific to the BrdU dose and administration schedule. Furthermore, we are not able to account for cells that incorporated BrdU but either migrated away from the SVZ (Kernie and Parent 2010) or were eliminated due to cell death (Winner et al. 2002).

Binge alcohol combined with TBI decreased neuroblasts in the SVZ and the RMS

We found that binge alcohol significantly decreased the number of neuroblasts in the SVZ 6 weeks after TBI. Although this decrease in BrdU⁺/DCX⁺ double-labeled cells was found bilaterally, it was much more pronounced in the ipsilesional SVZ. It is striking that our short binge alcohol administration had such a long-term effect on neuroblast number post-TBI. It is possible that binge alcohol changed the expression levels of trophic factors in the brain, for instance, BDNF, GDNF and EGF are all important for neurogenesis and are affected by alcohol (Janak et al. 2006). Additionally, it is possible that alcohol is affecting neurogenesis via epigenetic mechanisms (see Berkel and Pandey 2017 for review). One study found reduced methylation of the CpG (Cytosin-phosphatidyl-Guanin) of the promoter of NGF in the serum of alcohol dependent patients (Heberlein et al. 2013). Furthermore, alcohol was found to affect BDNF regulation by epigenetic mechanisms in mice (Stragier et al. 2015).

As we previously reported, using a repeated binge alcohol and TBI paradigm, alcohol administered rats had worse functional recovery on the sensitive skilled forelimb reaching task (Vaagenes et al. 2015). This task is known to be strongly dependent upon a well-functioning olfactory system (Whishaw and Tomie 1989), which is in turn modulated by continuous adult SVZ neurogenesis (Lim and Alvarez-Buylla 2016). Therefore, it is possible that the reduction in this SVZ early proliferative response and the subsequent decrease in the neuroblasts number as seen in the present study could be the reason underlying the observed decrease in functional recovery post binge alcohol/TBI (Vaagenes et al. 2015). In support of our work, another group has recently shown that SVZ neurogenesis was critical to functional recovery on the skilled forelimb reaching task (Shiromoto et al. 2017).

In conclusion, our results show that TBI alone robustly stimulates SVZ proliferation both short and long term in the adult male rat and that binge alcohol given prior to TBI dampens this proliferative process. Furthermore, binge alcohol given prior to TBI reduced the number of neuroblasts in the SVZ and those migrating in the RMS. Important to public health, a better understanding of these mechanisms could lead to better targeted therapies to improve functional recovery after TBI complicated by alcohol intoxication.

Table 2:

Summary of antibodies used for immunofluorescence and immunohistochemistry.

Primary Antibodies	Target/Antigen	Antigen Species	Immunogen	Source	Dilution
Rabbit anti-GFAP polyclonal	Astrocytes glial fibrillary acidic protein intermediate filament	Cow	Spinal cord GFAP	Dako Z0334 [RRID: AB_10013382	1:1000
Rabbit anti-doublecortin (DCX) polyclonal	Neuroblast microtubule associated protein	Synthetic	peptide of human DCX	Cell Signaling 4604S [RRID: AB_10693771]	1:500
Rabbit anti-Ibal Polyclonal	Macrophage/microglia calcium binding protein	Synthetic	Peptide of C-terminus of Ibal	Wako 019-19741 [RRID: AB_839503]	1:5000
Mouse IgG2a anti-BrdU monoclonal	5-Bromo-2-deoxyuridine (BrdU)	Synthetic	5-iodouridine covalently coupled to ovalbumin	Pierce MA3-071 [RRID: AB_10986341]	1:1000-4000
Mouse IgG1 anti-NeuN monoclonal	DNA-binding, neuron-specific protein NeuN	Mouse	Purified cell nuclei from mouse brain	Chemicon MAB377 [RRID: AB_2298772]	1:1000
Rabbit anti-Sox2 polyclonal	Transcription factor SRY (sex determining region Y)-box 2 expressed in neural stem cells	Synthetic	peptide conjugated to KLH derived from within residues 300 to the C-terminus of Human SOX2	Abcam Ab97959 [RRID: AB_10013822]	1:1000
Secondary Antibodies
Goat anti-mouse (AlexaFluor 488) polyclonal	Mouse IgG	Mouse	Gamma Immunoglobins Heavy and Light chains	ThermoFisher A11001 [RRID: AB_10566289]	1:500
Goat anti-mouse IgG2a (AlexaFluor 488) polyclonal	Mouse IgG2a	Mouse	IgG2a	ThermoFisher A21131 [RRID: AB_141618]	1:500
Goat anti-mouse IgG2a (biotinylated) polyclonal	Mouse IgG, Fcγ subclass 2a specific	Mouse	IgG2a	Jackson Immunoresearch 115-065-206 [RRID: AB_2338572]	1:500
Goat Anti-Rabbit (AP-conjugated) Polyclonal	Rabbit IgG	Rabbit	Gamma Immunoglobins Heavy and Light chains	Invitrogen 31340, RRID: AB_228339	1:500
Goat anti-rabbit (AlexaFluor 568) polyclonal	Rabbit IgG	Rabbit	Gamma Immunoglobins Heavy and Light chains	ThermoFisher A11036 [RRID: AB_143011]	1:500

Significance.

Binge alcohol drinking is a growing problem in America and is the most common mode of excess alcohol consumption. Patients with traumatic brain injury frequently also suffer from alcohol intoxication. Yet, how alcohol at the time of injury affects the brain reparative process is not well established. The present study provides evidence that a binge pattern of alcohol administration can reduce injury-induced neurogenesis in the adult male rat brain. Therefore, the effects of a relatively short binge of alcohol on the regenerative capacity of the brain may have major consequences for public health.