Regenerative Reponses of the Subventricular Zone Following Pediatric Traumatic Brain Injury

Pediatric Traumatic Brain Injury (TBI) is a significant problem that affects many children each year. Progress is being made in developing neuroprotective strategies to combat such injuries; however, investigators are a long way from therapies to fully preserve injured neurons and glia. To restore neurological function, regenerative strategies will be required. The Subventricular Zone (SVZ) harbors dividing cells that have the potential to regenerate multiple types of brain cells after injury; therefore, we evaluated regenerative responses of the SVZ after pediatric. We used controlled cortical impact (CCI) injury to produced comparable damage to the somatosensory cortex of rats at postnatal day 6 (P6), P11, P17 and P60 and mice at P14. At both 48 and 96 hours after injury, the mitotic indices of animals injured at pediatric ages were significantly increased vs. sham operated and naïve controls and the regenerative response was more robust in the immature vs. the adult brain. A 4-marker flow cytometry panel and immunolabeling for Nestin/Ki-67/Mash1 showed increases in NSCs as well as in 2 classes of multipotential progenitors. BrdU+/DCX+ cells were increased in the ipsilateral SVZ and parenchyma adjacent to the lesion 14 days after rat CCI. However, very few new mature neurons were seen in the lesion 28 days after injury. Altogether, our data indicate that although the immature brain is capable of mounting a robust proliferative response to CCI that includes an expansion of primitive NSCs and certain progenitors, these responses do not result in sustained neurogenesis or significant neuronal replacement. Introduction Pediatric Traumatic Brain Injury (TBI) is a significant problem that affects 600,000 children under the age of 14 each year. Approximately 60,000 of these children require hospitalization and estimates are that 7,400 children die each year from TBI-related injuries. The estimated financial burden of pediatric TBI is $1 billion/year in hospital costs alone. Given this enormous financial burden in addition to the emotional problem placed on caregivers, there is strong rationale for additional studies of pediatric TBI. Due to the fact that the young nervous system possesses a greater ability to recover from injury than the adult nervous system in general and also in response to mechanical brain injuries, it is informative to study those properties of the immature brain that allows for greater recovery. The Subventricular Zone (SVZ) is a mosaic of neural precursors that includes multipotential neural stem cells (NSCs), bipotential progenitors and unipotential progenitors. Virtually all of the neurons and glia that populate the mature brain arise from neural precursors (NPs) that reside in germinal zones immediately adjacent to the lateral ventricles during development. The SVZ achieves its maximal size and heterogeneity during the neonatal period when the brain is in a dynamic state of development. With the recent discovery that adult olfactory bulb neurogenesis in the human brain likely ceases after 18 months of age, there is increased interest in studying the neural precursors (NPs) of the neonate. Changes in SVZ cell proliferation have been extensively examined across a number of adult animal models of traumatic brain injury including penetrating brain injuries, aspiration lesions, fluid percussion injury and controlled cortical impact (CCI). Studies to date have revealed injury model dependent differences in the SVZ proliferative responses. In rats, the total number of cells in the adult SVZ increases 1 week following aspiration lesions of the somatosensory cerebral cortex. In the fluid percussion injury model in adult rats, the number of 5-bromo-2-deoxyuridine (BrdU)-labeled SVZ cells increases 2 and 4 days after injury. In the CCI model, short term increases in proliferation were seen in adult rats 14 The dorsal aspect of the ipsilateral SVZ to the injury is the area of the SVZ most involved in proliferation following injury and has also been characterized in the adult mouse after CCI. Therefore, it has been well established that the adult SVZ is capable of mounting a robust proliferative response to a variety of mechanical traumatic brain insults. Whereas cells born in the ventricular zone and SVZ of the developing forebrain migrate towards many nuclei, neuroblasts in the intact adult SVZ of most vertebrates migrate primarily to the olfactory bulb along the rostral and medial migratory streams. 16 While this appears to be the case in the intact brain, a number of studies indicate that brain injury can induce SVZ cells to migrate more broadly, especially towards the lesion. Using BrdU labeling, newly generated neurons can be followed and shown to express both early markers, such as Doublecortin (Dcx) as well as markers of more mature neurons such as Class III beta tubulin, calretinin or NeuN. Additional studies have been performed using retrograde labeling to provide evidence that these new neurons are indeed integrated into neural circuits. In our studies of neonatal hypoxicischemic brain injury, we have documented the significant production of new neurons from the SVZ that begins a few days after the injury and persists for at least 2 months. 19 To date, no studies have examined if similar increases in SVZ proliferation and regeneration occur following focal, mechanical traumatic brain injury in pediatric-aged rodents. As studies of traumatic injury in adult rats have rarely seen migration of SVZ cells into the neocortex, we hypothesized that the production of new neocortical neurons would be greater in the immature brain. We recently established a novel flow cytometry protocol to quantify the relative proportions of 8 unique SVZ progenitors as well as NSCs. This powerful tool allows us to examine the effects of injury and other genetic manipulations on SVZ cells with a greater level of resolution previously unattainable. We have applied this method to compare the composition of SVZ cells between wild-type and genetically altered mice, as well as in a mouse model of hypoxia-ischemia. In the studies reported here, we used several complementary approaches to evaluate changes in the SVZ NSC and progenitor responses subsequent to pediatric TBI. We used CCI to equivalently damage the somatosensory cortex of rats at postnatal day 6 (P6), P11 and P17 and P60. The P6 time point was chosen to allow a direct comparison with our studies of hypoxic-ischemic injury; the P11 animal was chosen to model injury to neonates, the P21 rat was chosen to model traumatic injury in toddlers and the P60 rat was chosen to model injury in adults. In addition, we modified the CCI model to produce a similar injury in P14 mouse. Using these injury paradigms, we evaluated cell proliferation within the SVZ following CCI, characterized which populations were changing following injury and compared the regenerative potential across this range of ages. These studies reveal that the developing brain mounts a much more robust proliferative and regenerative response to focal TBI than the adult brain, which arises from the expansion of NSCs and specific subsets of progenitors in the SVZ. Materials and Methods Reagents Common laboratory chemicals were purchased from either Sigma (Sigma, St. Louis, Mo., USA) or VWR International (Radnor, PA). Cell culture media were purchased from either Invitrogen or Sigma. Recombinant EGF and FGF2 were purchased from PeproTech, Rocky Hill, NJ, USA. Optimal cutting temperature compound embedding medium was purchased from Sakura Finetek, Torrance, CA, USA. The following antibodies were used for our studies: Rat anti-BrdU (1/30, Accurate Chemicals, Westbury, N.Y., USA); mouse anti-Mash1 (BD Pharmingen San Jose, CA, USA, www.bdbiosciences.com; 1:50); chicken anti-Nestin (Aves Labs Inc. Tigard, OR, USA, www.aveslab.com; 1:250); rabbit anti-Ki-67 (Vector, Burlingame, CA, USA, www.vectorlabs.com; Z0311, 1:500); mouse anti-NeuN (Millipore/Chemicon, Billerica, MA, USA, www.chemicon.com; 1:100), mouse anti-HuC/D (Invitrogen, Grand Island, NY, USA, www.invitrogen.com; 1:50). The following secondary antibodies were used: RedX-conjugated donkey anti-rat; Alexa 488 conjugated-donkey anti-rabbit, Alexa 549-conjugated donkey antichicken and AMCA-conjugated donkey anti-mouse (1/200, all from Jackson ImmunoResearch, West Grove, Pa., USA). 4’,6’-diamidino-2-phenylindole (DAPI) was purchased from Sigma. Fluoro-Gel was purchased from Electron Microscopy Services, Hatfield, PA, USA. For surface marker analysis by flow cytometry the following antibodies were used: antibodies against Lewis-X-Alexa Flour 488 (1:20, LeX/CD15, MMA; BD Bioscience, San Diego, CA), CD133-APC (1:50,13A4; eBioscience, San Diego, CA), CD140a-PE (1:400, APA5; BioLegend) and NG2 Chondroitin Sulfate Proteoglycan (1:100, AB5320; Millipore); goat antirabbit IgG Alexa Fluor 700 (1:100; Invitrogen). Ultrapure formaldehyde was purchased from Polysciences, Inc; Warrington, PA. Controlled Cortical Impact All experiments were performed in accordance with protocols approved by the institutional animal care and use committee of the University of Rutgers-New Jersey Medical School. Timed pregnant Sprague Dawley rats (Charles River, Wilmington, Del., USA) and timed pregnant C57Bl/6 mice were housed and cared for by the Department of Comparative Medicine. Unilateral CCI was performed on the somatosensory cortex of postnatal day 6 (P6), P11, P17 and P60 rat pups and on P14 mouse pups. The pups were anesthetized with ketamine/xylazine. Once fully anesthetized, the scalp was cleansed and an incision along the midline created to expose the skull. In order to produce equivalent injuries among different aged animals and across species, different size impactor tips and depths of injuries were performed (Table 1). Age-matched sham-operated animals only received craniectomies. Animals were placed on heating pads at 37° and monitored continuously for 2 h after surgery. In addition, immediately after surgery, all animals received 3% body weight of 0.9% saline subcutaneously (SC) to prevent dehydration. P60 rats were singly caged until they were sacrificed. All other rat age groups and the P14 mouse group were returned to their dam cages to resume nursing with their mothers until they were sacrificed. BrdU Injections and Intracardiac Perfusions BrdU was administered intraperitoneal at 50 mg/kg dissolved in 0.007 N NaOH in PBS. Several dosage paradigms were used as described later. For intracardiac perfusions, animals were anesthetized with a mixture of ketamine (75 mg/kg) and xylazine (5 mg/kg) and then fixed by intracardiac perfusion with media followed by 3% paraformaldehyde in PBS 4 hours following the final BrdU injection. Each brain was removed from the skull, post-fixed overnight and then cryoprotected for 24 h in 30% sucrose in water. The brains were frozen in optimal cutting temperature compound embedding medium on a dry-ice/ethanol slush. Immunofluorescence Immunofluorescence was performed on 40 μm sections as previously described. After secondary antibody incubation the sections were washed, counterstained with 1 μg/ml DAPI for 5-10 minutes, and coverslipped with Fluoro-Gel. The sections were analyzed using an Olympus AX70 fluorescence microscope equipped with a Q-imaging CCD camera and IPLab imaging software. All secondary antibody combinations were carefully examined to ensure that there was no bleed through between fluorescent dyes or cross-reactivity between secondary antibodies. No signal above background was obtained when the primary antibodies were replaced with pre-immune sera. Quantification of Proliferation To quantify BrdU labeled cells to obtain a mitotic index, six sections were sampled at 150 μm intervals per brain. The dorsolateral SVZ was divided into three distinct anatomical subregions as previously described. Briefly, the medial region consists of the densely packed SVZ cells immediately adjacent to the ependymal layer. The mediolateral region is approximately 200 μm lateral from the medial region and is much less dense than the medial region. Finally, the lateral SVZ is 200 μm lateral to the mediolateral region and consists of densely packed cells. Two fields per SVZ region were assessed per section (ipsilateral and contralateral to the injury site). Each field occupied an area of 50μm. The numbers of BrdU+ and total DAPI+ cells in each region of the damaged neocortex were then quantified and averaged. The data are presented as the percentage of labeled cells per field after correction using the Abercrombie correction as described in greater detail in a previous publication. For stereological evaluation of triple Immunofluorescence labeled specimens, 40 μm sections were analyzed using the MicroBrightfield StereoInvestigator program on an Olympus BX51 microscope. 23 2222 Six sections were sampled at 150 μm intervals per brain. For each section, quantification of Nestin+/Ki-67+/Mash1stem cells and Nestin+/Ki-67+/Mash1+ intermediate progenitors was conducted in the medial, mediolateral and lateral subregions of the SVZ as described above. Six fields of 50 μm were assessed per section in the ipsilateral and contralateral SVZ. The numbers of Nestin+/Ki-67+/Mash1and Nestin+/Ki-67+/Mash1+ cells in each region of the damaged neocortex were then quantified, averaged and then converted to cells/mm. Neurosphere Propagation and Quantification Neural stem cells and neural precursors from the SVZ of CCI, sham operated and untouched treated animals were harvested and grown in culture as neurospheres. Two days after CCI injury to the somatosensory cortex was performed on male Sprague Dawley rats at P11. The animals were sacrificed by decapitation and their brains immediately removed for SVZ dissection as previously described . Briefly, a 3 mm thick coronal section of the brain was obtained by cutting 2mm and 5mm from the rostral end of the brain and placed in cold PGM [(PBS, 1mM MgCl2 and 0.6% dextrose in PBS). The hippocampus, the corpus callosum and meninges were removed and a wedge of brain tissue between 1 o’clock and 3 o’clock enclosing the cortex and ventricle containing the SVZ was excised. The tissue was digested enzymatically with a mixture containing 0.25% trypsin/EDTA, 100 units Papain, 250 μg/ml of DNase I and 3 mM MgSO4 in DMEM/F12. The reaction was stopped with trypsin inhibitor in chemically defined medium ProN [DMEM/F12, B27 supplement, 50 ug/ml apotransferrin and 50 ug/ml gentamicin (50 ug/ml)]. The tissue was triturated to obtain a single cell suspension, collected by centrifugation and counted with a hemocytometer. Dissociated cells were seeded in 12-well culture plates at a density of 5x10 cells/mL in ProN media containing 20 ng/ml EGF and 10 ng/ml FGF–2. Cultures were fed every 2 days by replacing half of the media with an equal volume of fresh media. Neurosphere numbers were quantified after 7 days in vitro as described previously. Briefly, six random fields per well in quadruplicate were imaged in phase contrast with a 10x objective. Neurospheres of at least 25 μm in diameter were counted and a total of 24 photos per condition were analyzed. Neurosphere diameters were determined from the same images using a microscale with 10 μm subunits as reference. Flow Cytometry C57BL/6 pups at 2 days recovery after CCI (P16) were decapitated and the contralateral and ipsilateral SVZs from injured brains, as well as SVZs from sham-operated animals and untouched control animals were isolated by microdissection and placed in dishes containing cold PGM. SVZs were pooled and incubated with 0.45 Wünsch unit /ml of Liberase DH (Roche) and 250 μg of DNase1 (Sigma) in PGM and were shaken at 230 rpms (Innova 2300, New Brunswick Scientific, Edison, NJ) at 37°C for 30 min. Enzymatic digestions were quenched with 10 ml of PGB (PBS without Mg and Ca with 0.6% dextrose and 2 mg/ml fraction V of BSAFisher) and cells were centrifuged for 5 min at 200 xg. Cells were dissociated by repeated trituration, collected by centrifugation, counted with a hemocytometer and diluted to at least 10 cells per 50 μl of PGB. All staining was performed in 96 V-bottom plates using 150 μl volume. For surface marker analysis, cells were incubated in PGB for 25 min with antibodies against Lewis-X, CD133-APC, CD140a, and NG2 Chondroitin Sulfate Proteoglycan. Cells were washed with PGB by centrifugation at 278 xg. Secondary antibody Goat anti-rabbit IgG Alexa Fluor 700 was used to detect NG2. Cells were incubated with secondary antibodies and DAPI (1:3000, Invitrogen) in PGB for 20 min and then washed by centrifugation in PGB at 278 x g. Cells from SVZ were fixed with 1% ultrapure formaldehyde for 20 min, collected by centrifugation for 9 min at 609 x g, resuspended in PGB and stored at 4°C overnight. All sample data were collected on a BD LSR II (BD Biosciences Immunocytometry Systems). Matching isotype controls for all antibodies were used and gates were set based on these isotype controls. Data were analyzed using FlowJo software (Tree Star, Inc; Ashland, OR). Statistical Analyses Results from the cell counts were analyzed for statistical significance using ANOVA followed by Tukey’s post hoc test. All data are presented as means ± SEM. Comparisons were interpreted as significant when associated with p < 0.05.