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Regional distribution of Fluoro-Jade B staining in the hippocampus following traumatic brain injury
Experimental Neurology 193 (2005) 125 – 130 www.elsevier.com/locate/yexnr
Regional distribution of Fluoro-Jade B staining in the hippocampus following traumatic brain injury Kevin J. Andersona,b,*, Kelly M. Millera, Isabella Fugacciaa, Stephen W. Scheff a a
Sanders-Brown Center on Aging and Department of Anatomy and Neurobiology, University of Kentucky, Lexington, KY, 40536, USA b Department of Physiological Sciences, University of Florida, Gainesville, FL 32610, USA Received 24 May 2004; revised 18 November 2004; accepted 30 November 2004
Abstract Fluoro-Jade B (FJB) is an anionic fluorescein derivative that has been reported to specifically stain degenerating neurons. We were interested in applying FJB staining in a well-characterized model of traumatic brain injury (TBI) in order to estimate the total number of neurons in different regions of the hippocampus that die after a mild or moderate injury. Rats were subjected to a mild or moderate unilateral cortical contusion (1.0- or 1.5-mm displacement from the cortical surface) with a controlled cortical impacting device. Animals were allowed to survive for 1, 2, or 7 days and the total number of FJB-positive neurons in hippocampal areas CA1, CA3, and the dentate gyrus granule layer was estimated using sterological methods. The region that had the highest number of FJP-positive neurons after TBI was the dentate gyrus. In both 1- and 1.5-mm injuries, FJB-positive granule cells were observed throughout the rostro-caudal extent of the dentate. In contrast, labeled pyramidal neurons of area CA3 were most numerous after the 1.5-mm injury. The area that had the fewest number of FJBlabeled cells was area CA1 with only scattered neurons seen in the 1.5-mm group. In both injury groups and in all hippocampal regions, more FJB-positive neurons were seen at the earlier times post injury (1 and 2 days) than at 7 days. FJB appears to be a reliable marker for neuronal vulnerability following TBI. D 2004 Elsevier Inc. All rights reserved. Keywords: Cell death; Cortical contusion; Fluoro-Jade; Hippocampus; Stereology
Introduction Rodent models of cortical impact injury produces spatial learning and memory deficits, similar to those seen in human traumatic brain injury, and it is thought that hippocampal cell death may contribute to these deficits (Graham et al., 2000; Maxwell et al., 2003). In the hippocampus, experimental traumatic brain injury (TBI) using either controlled cortical impact or fluid percussion produces damage and death of pyramidal neurons throughout Ammon’s horn (Baldwin et al., 1997; Colicos and Dash, 1996; Conti et al., 1998; Grady et al., 2003; Hallam et al., * Corresponding author. Department of Physiological Sciences, PO Box 100144, University of Florida, Gainesville, FL 32610, USA. Fax: +1 352 392 5145. E-mail address: [email protected] (K.J. Anderson). 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.11.025
2004; McCullers et al., 2002; Mitchell et al., 2002; Miyazaki et al., 1992; Reeves et al., 1995; Sato et al., 2001). This loss of neurons may be correlated with impaired performance in learning and memory tasks seen following these types of insults (Hallam et al., 2004; Hamm et al., 1992; Smith et al., 1991). Fluoro-Jade B (FJB) is an anionic fluorescein derivative that has been reported to specifically stain degenerating neurons (Schmued and Hopkins, 2000a,b). Several models of experimentally induced neuronal injury, including kainic acid, binge alcohol consumption, and trimethyltin, have been used to determine that Fluoro-Jade and FJB are specific markers for neurons that are in the process of degenerating in the rat brain (Hopkins et al., 2000; Schmued and Hopkins, 2000a,b, Obernier et al., 2002) and shows a high correlation with other traditional markers of neuronal degeneration such as silver stains (Schmued
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et al., 1997). Other studies have used FJB as a marker of neuronal degeneration in both controlled cortical impact and fluid percussion models of TBI (Hallam et al., 2004; Lyeth et al., 2001; Sato et al., 2001; Zhao et al., 2003; Zwienenberg et al., 2001). Stereological techniques have been used to show a loss of pyramidal neurons in hippocampal CA1, CA3, and the hilus 14 days after both fluid percussion injury and controlled cortical impact (Baldwin et al., 1997; Grady et al., 2003; McCullers et al., 2002). However, it is unknown whether the severity of cortical injury correlates with the number of dying hippocampal neurons and whether there are differences in the total number of dying neurons between the hippocampal subregions following a cortical contusion. In this study, we have used a model of controlled cortical impact and examined hippocampal pyramidal and granule neurons using FJB as a marker of neuronal death in order to test the following hypotheses: (1) the number of degenerating neurons correlates with injury severity, and (2) there are more CA3 pyramidal neurons lost after this type of insult.
Materials and methods Head trauma Young-adult male Sprague–Dawley rats (Harlan; 200– 225 g) were subjected to a mild or moderate unilateral cortical contusion (1 or 1.5 mm, respectively) as previously described (Scheff and Sullivan, 1999; Sullivan et al., 2002; Sullivan et al., 1998). The moderate cortical contusion used in these experiments results in severe behavioral deficits, significant loss of cortical tissue, blood–brain barrier disruption, and loss of hippocampal neurons (Baldwin and Scheff, 1996; Baldwin et al., 1997; Beer et al., 2000; Scheff et al., 1997, Sullivan et al., 1998), mimicking the sequelae of human closed-head injury. All subjects were anesthetized with isoflurane (2%) and placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA) prior to TBI. Using sterile procedures, the skin was retracted, and a point was identified midway between bregma and lambda and midway between the central suture and the temporalis muscle laterally. This location, which was consistent between all animals used in this study, constituted the central location of the 6 mm in diameter circular craniotomy that was made with a handheld Michele trephine (Miltex, York, PA). The skull cap was carefully removed without disruption of the underlying dura. Prior to the injury, the head of the animal was angled in a medial to lateral plane so that the impacting tip was perpendicular to the exposed cortical surface. This was accomplished by rotating the entire stereotaxic frame in the transverse plane while leaving the nose bar at 5.0. The exposed brain was injured using a pneumatically controlled impacting device (PSI, Fairfax, VA) with a 5-mm beveled tip, which compressed the
cortex at 3.5 m/s to a depth of either 1 or 1.5 mm (Baldwin and Scheff, 1996; Sullivan et al., 2000). Following injury, Surgicel (Johnson and Johnson, Arlington, TX) was laid upon the dura and the skull cap was replaced. A thin coat of dental acrylic was then spread over the craniotomy site and allowed to dry before the wound was stapled closed. During all surgical procedures and recovery, the core body temperature of the animals was maintained at 36–378C using heating pads. Animals were killed at 1 (1 mm, n = 4; 1.5 mm, n = 5), 2 (1 mm, n = 5; 1.5 mm, n = 5), or 7 (1 mm, n = 5; 1.5 mm, n = 5) days post injury for histological analysis. The Animal Care and Use Committee at the University of Kentucky approved all animal procedures. All efforts were made to minimize both the possible suffering and number of animals used. Tissue processing Rats were overdosed with sodium pentobarbital and transcardially perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde (PF) in PBS. The brains were removed from the cranium and postfixed in PF for 24 h at 48C before cryoprotecting in 20% sucrose/PBS at 48C. Brains were frozen in powdered dry ice and serial 50-Am cryosections through the entire extent of the hippocampal formation were cut on a sliding microtome in the coronal plane, placed into PBS, and mounted onto Fisher Superfrost Plus slides. Slides were dried on a slide warmer for 1 h and then processed for FJB and concurrent 4V,6-diamidino-2-phenylindole (DAPI) histochemistry. Histology and immunohistochemistry Staining with FJB and DAPI was performed using slight modifications of the technique described by Schmued and Hopkins (2000a). Sections were first incubated in a solution of 1% NaOH in 80% ethanol for 5 min and then were hydrated in graded ethanols (75, 50 and 25%; 5 min each) and distilled water. They were then incubated in a solution of 0.06% potassium permanganate for 10 min on a rotating stage, rinsed in distilled water for 2 min and incubated in a 0.0004% solution of FJB (HistoChem Inc., Jefferson, AR) and 0.0004% DAPI (Sigma, St. Louis, MO) for 20 min. Sections were then rinsed in distilled water (3 2 min), air-dried then placed on a slide warmer until fully dry (5–10 min). The dry slides were cleared in CitriSolv (2 5 min) and coverslipped with DPX (Fluka). Stereology The total number of FJB-positive neurons following TBI was determined blindly with respect to both severity and days post injury. Following sectioning of the entire
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extent of the hippocampal formation, a minimum of 12 equally spaced sections, with a variable starting location, were assessed employing an image analysis system (Bioquant Nova Prime v6.50.10, Nashville, TN) interfaced with an Olympus BX50 microscope and an Optronics VI470 camera (Goleta, CA). The entire reference volume (Vref) of the individual hippocampal subregions (dentate gyrus granule cells, CA3 pyramidal cells, and CA1 pyramidal cells) was estimated using the Cavalieri method (Michel and Cruz-Orive, 1988) and the anatomical boundaries as described by Amaral and Witter (1995). These various cell layers were identified using UV excitation of DAPI-positive neurons. Since the total number of FJB-positive neurons on any given section within each of the defined subregions often appeared to occur in clusters and didn’t appear to exceed more than 150 neurons, every FJB-positive neuron was counted under blue (450–490 nm) excitation light, employing a 20 objective to verify that the cell was a neuron. In this regard, individual small dissectors, often employed in unbiased stereological counting schemes (Gundersen et al.,
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1988) of large populations was not necessary to obtain an estimate of the individual section volume density. Estimates of the total number of FJB-positive neurons was P determined with the formula N v = [ (N / V subregion) / n] d Vref where N v = total estimated number of FBJ positive neurons in a subregion; N = total number of FJB-positive neurons counted in a subregion on an individual section; V subregion = volume of subregion on an individual section; n = number of individual sections analyzed; Vref = total reference volume for the subregion. Comparison of NeuroSilver and FJB staining An additional cohort of animals to those described above received a 1.5-mm contusion and were killed at 2 days post injury (n = 6). Alternate sections from these animals were processed for FJB and NeuroSilver histology (FD Neuro Technologies Inc., Baltimore, MD) and the total number of FJB-positive and NeuroSilver-positive neurons in the dentate gyrus were estimated using the same stereological techniques as described above.
Fig. 1. Fluoro-Jade B fluorescent neurons in the hippocampus following TBI. These are sections from animals that were sacrificed at 1 day post injury. (A) Area CA1 from an animal with a 1.5-mm injury; (B) Area CA1 from an animal with a 1-mm injury. (C) Area CA3 from an animal with a 1.5-mm injury; (D) Area CA3 from an animal with a 1-mm injury. (E) Dentate gyrus and hilus from an animal with a 1.5-mm injury; (F) Dentate gyrus and hilus from an animal with a 1-mm injury. Note the higher number of Fluoro-Jade positive neurons in all regions of animals that had received a 1.5-mm injury. Abbreviations: Pyr = Pyramidal cell layer; SG = Stratum granulosum of the dentate gyrus; Hi = Hilus of the dentate gyrus. Calibration bar = 100 Am.
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Data analysis The collected data were analyzed using a multi-factor analysis of variance with injury severity and days post injury used as main factors and subregions of the hippocampus used as a repeated measure within subject factor. For post hoc comparisons, the Tukey/Kramer test was used with significance set at P b 0.05. For comparison of FJBand NeuroSilver-stained neurons, a paired t test was used with significance also set at P b 0.05.
Results In all animals injured at both the mild and moderate level of TBI, there were obvious FJB-positive neurons in the hippocampus (Fig. 1). The statistical analysis revealed a significantly greater number of FJB-positive neurons [F = 17.059 df 1,46 P b 0.0004] in the hippocampal formation of moderately injured animals compared to the mild group. The analysis revealed a difference in days post injury [F = 4.210 df 2,46 P b 0.03] and as shown in Fig. 2, the greatest number of FJB-positive neurons was observed at 1 day post injury. The analysis revealed a significant regional distribution in FJB-positive neurons following the injury [F = 56.386 df 2,46 P b 0.0001]. A post hoc analysis revealed that the dentate gyrus granule cell layer had significantly more FJB-positive neurons than both the CA3 and CA1 regions and the CA3 region had significantly more positive neurons than CA1.
Fig. 2. Estimates of the total number of Fluoro-Jade B positive neurons in different regions of the hippocampus following either a mild or moderate cortical contusion. As early as 1 day following the injury, significant numbers of positive neurons were observed in the dentate gyrus granule cell layer. This number significantly increased with a moderate level of injury. The CA1 pyramidal cells showed the least sensitivity to the injury but did have significant number of positive cells with the moderate injury. The early time points post injury demonstrated the greatest numbers of FJBpositive cells. Bars represent means F SEM. Asterisks represent a significant difference from the 1-day post injury group as determined by Tukey/Kramer post hoc analysis ( P b 0.05).
Differences were further probed by analyzing changes in FBJ staining in mild and moderate injury separately. The analysis revealed no significant effect of days post injury for the mild injury severity [F = 0.580 df 2,22 P N 0.1] but did reveal a significant change in the regional distribution of FJB staining [F = 18.839 df 2,22 P b 0.0001] with the most significant differences between the dentate gyrus and hippocampal regions CA3 and CA1 as revealed by the post hoc analysis. CA3 did not differ significantly from CA1. Analysis of the moderately injured group demonstrated a significant days post injury effect [F = 4.307 df 2,24 P b 0.05] with post hoc analysis demonstrating significant differences between 1-day post injury and the 2- and 7-day post injury groups (Fig. 2). As with the mild injury group, there was a significant regional difference in the FJB staining in the moderate injured group [F = 46.635 df 2,24 P b 0.0001]. Post hoc analysis demonstrated that the dentate gyrus region had significantly more positive neurons than both CA3 and CA1 and that there were more positive CA3 neurons compared to area CA1. In order to compare FJB staining with NeuroSilver staining, we estimated the total number of positive neurons in the dentate gyrus at 2 days post injury (1.5 mm). There was no significant difference between the number of FJBpositive and NeuroSilver-positive neurons (FJB = 4146 F 443; NeuroSilver = 4746 F 916; P N 0.05) (Fig. 3).
Discussion In this study, we have demonstrated a regional difference in FJB staining among hippocampal subregions using a controlled cortical impact TBI model. Using both a mild and moderate level of injury, we demonstrated greater numbers of FJB-positive neurons as a function of increased injury severity. The dentate gyrus granule layer displayed the greatest number of injured neurons regardless of the level of severity with significant numbers of positive cells observed as early as 24 h following a mild injury. In addition, area CA3 showed significantly more FJB-positive neurons than area CA1 in the moderately injured group. This is the first quantitative report of extensive hippocampal granule cell injury following a cortical contusion model of TBI. Two previous reports employing a lateral fluid percussion (FP) model demonstrated FJB-positive granule cells but the number of cells was not quantified (Hallam et al., 2004; Sato et al., 2001). Similarly, two papers have reported on the time course and number of FJB-positive CA3 neurons following FP injury but did not estimate the total number of these neurons (Zhao et al., 2003; Zwienenberg et al., 2001). The present results coupled with the previously published findings support the idea that these hippocampal structures are sensitive to TBI. Previous studies have highlighted significant neuronal loss in the CA1 and CA3 regions of the hippocampus following a controlled cortical impact model (Baldwin et al., 1997; McCullers et al., 2002). A recent FP study has also
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Fig. 3. Photomicrographs of adjacent sections stained with NeuroSilver (A) and Fluoro-Jade B (B) in an animal subjected to a 1.5-mm injury and sacrificed 2 days post injury. Note the nearly identical pattern of staining in the stratum granulosum of the dentate gyrus (SG). Calibration bar = 100 Am.
demonstrated that, following trauma, the total number of hilar CA3 neurons as determined by stereology is decreased when examined 2 weeks after the injury (Grady et al., 2003). However, no significant differences were seen in the number of dentate gyrus granule neurons or hippocampal area CA1 pyramidal neurons as a function of injury. In our study, the largest number of FJB-positive neurons were seen in the granule cell layer of the dentate gyrus. However, estimates of the total number of granule cells range from 750,000 to 1,550,000 (Grady et al., 2003; Madeira et al., 1988; McCullers et al., 2002; Rapp and Gallagher, 1996; Rasmussen et al., 1996; West et al., 1991; Xavier et al., 1999). Thus, the proportion of neurons dying in the hippocampal dentate gyrus may be smaller than area CA3 (including the hilus) where the total number of neurons have been estimated to be between 267,600 and 400,000 (Grady et al., 2003; Rasmussen et al., 1996; West et al., 1991). Similarly, although few FJB-positive neurons were seen in area CA1, the total number of CA1 pyramidal neurons is similar to area CA3; estimated to be between 245,000 to 570,000 (Grady et al., 2003; McCullers et al., 2002; Rapp and Gallagher, 1996; Rasmussen et al., 1996; West et al., 1991; Xavier et al., 1999). Therefore, when
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described as a proportion of the total number of neurons, areas CA3 and CA1 may actually be impacted more than the granule cell layer of the dentate gyrus. While the total number of granule cells lost may be very small relative to the total number of neurons, this still may contribute to the behavioral and neurological deficits following TBI. As noted earlier, FJB appears to specifically stain degenerating neurons. We have also shown that FJB staining correlates very closely with more traditional markers of neuronal degeneration (NeuroSilver) in TBI. The advantage to using FJB lies in its ease of use, consistency, and that fact that is less capricious than silver methods. We have recently shown that while FJB stains dead or dying neurons following TBI, it does not label dying neurons in the spinal cord following a contusion injury (Anderson et al., 2003). The identity of the binding target of FJB is still unknown, although it is hypothesized that since FJB is a poly-anionic fluorescein derivative, it may label cationic products of degeneration such as spermidine, cadaverine, or putrescine (Schmued and Hopkins, 2000a). Although FJB does not appear to be a useful tool to study neuronal death in all models of neurotrauma, it does appear to be valuable in examining neuronal death in models of TBI (Anderson et al., 2003). FJB-positive neurons have been observed in the hippocampus following FP injury as early as 3 h after the insult with a peak at 1 day (Sato et al., 2001). These findings are similar to the findings of the current study where the peak number of neurons is generally seen at 1 day post injury. In addition, FJB-positive neurons appear to be greatly diminished by 7 days post injury following both FP (Sato et al., 2001) and cortical contusion (Fig. 2, present study). It is unclear at present if the 1-day post injury time point following a cortical contusion model of TBI represents the maximum FJB staining and thus it will be of interest to assess earlier time points post injury. Such an investigation may increase our understanding of the critical window for therapeutic intervention. In summary, FJB appears to give a bsnapshotQ of neurons that are dead or dying following cerebral injury and thus may be a reliable marker to test neuroprotective agents following TBI.
Acknowledgments This study was supported by the Kentucky Spinal Cord and Head Injury Research Trust #7–18, the Spinal Cord Research Foundation, and the NIH (NS 39828, AG21981). The authors gratefully acknowledge the technical assistance of Kelly Roberts.
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Regional distribution of Fluoro-Jade B staining in the hippocampus following traumatic brain injury Kevin J. Andersona,b,*, Kelly M. Millera, Isabella Fugacciaa, Stephen W. Scheff a a
Sanders-Brown Center on Aging and Department of Anatomy and Neurobiology, University of Kentucky, Lexington, KY, 40536, USA b Department of Physiological Sciences, University of Florida, Gainesville, FL 32610, USA Received 24 May 2004; revised 18 November 2004; accepted 30 November 2004
Abstract Fluoro-Jade B (FJB) is an anionic fluorescein derivative that has been reported to specifically stain degenerating neurons. We were interested in applying FJB staining in a well-characterized model of traumatic brain injury (TBI) in order to estimate the total number of neurons in different regions of the hippocampus that die after a mild or moderate injury. Rats were subjected to a mild or moderate unilateral cortical contusion (1.0- or 1.5-mm displacement from the cortical surface) with a controlled cortical impacting device. Animals were allowed to survive for 1, 2, or 7 days and the total number of FJB-positive neurons in hippocampal areas CA1, CA3, and the dentate gyrus granule layer was estimated using sterological methods. The region that had the highest number of FJP-positive neurons after TBI was the dentate gyrus. In both 1- and 1.5-mm injuries, FJB-positive granule cells were observed throughout the rostro-caudal extent of the dentate. In contrast, labeled pyramidal neurons of area CA3 were most numerous after the 1.5-mm injury. The area that had the fewest number of FJBlabeled cells was area CA1 with only scattered neurons seen in the 1.5-mm group. In both injury groups and in all hippocampal regions, more FJB-positive neurons were seen at the earlier times post injury (1 and 2 days) than at 7 days. FJB appears to be a reliable marker for neuronal vulnerability following TBI. D 2004 Elsevier Inc. All rights reserved. Keywords: Cell death; Cortical contusion; Fluoro-Jade; Hippocampus; Stereology
Introduction Rodent models of cortical impact injury produces spatial learning and memory deficits, similar to those seen in human traumatic brain injury, and it is thought that hippocampal cell death may contribute to these deficits (Graham et al., 2000; Maxwell et al., 2003). In the hippocampus, experimental traumatic brain injury (TBI) using either controlled cortical impact or fluid percussion produces damage and death of pyramidal neurons throughout Ammon’s horn (Baldwin et al., 1997; Colicos and Dash, 1996; Conti et al., 1998; Grady et al., 2003; Hallam et al., * Corresponding author. Department of Physiological Sciences, PO Box 100144, University of Florida, Gainesville, FL 32610, USA. Fax: +1 352 392 5145. E-mail address: [email protected] (K.J. Anderson). 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.11.025
2004; McCullers et al., 2002; Mitchell et al., 2002; Miyazaki et al., 1992; Reeves et al., 1995; Sato et al., 2001). This loss of neurons may be correlated with impaired performance in learning and memory tasks seen following these types of insults (Hallam et al., 2004; Hamm et al., 1992; Smith et al., 1991). Fluoro-Jade B (FJB) is an anionic fluorescein derivative that has been reported to specifically stain degenerating neurons (Schmued and Hopkins, 2000a,b). Several models of experimentally induced neuronal injury, including kainic acid, binge alcohol consumption, and trimethyltin, have been used to determine that Fluoro-Jade and FJB are specific markers for neurons that are in the process of degenerating in the rat brain (Hopkins et al., 2000; Schmued and Hopkins, 2000a,b, Obernier et al., 2002) and shows a high correlation with other traditional markers of neuronal degeneration such as silver stains (Schmued
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et al., 1997). Other studies have used FJB as a marker of neuronal degeneration in both controlled cortical impact and fluid percussion models of TBI (Hallam et al., 2004; Lyeth et al., 2001; Sato et al., 2001; Zhao et al., 2003; Zwienenberg et al., 2001). Stereological techniques have been used to show a loss of pyramidal neurons in hippocampal CA1, CA3, and the hilus 14 days after both fluid percussion injury and controlled cortical impact (Baldwin et al., 1997; Grady et al., 2003; McCullers et al., 2002). However, it is unknown whether the severity of cortical injury correlates with the number of dying hippocampal neurons and whether there are differences in the total number of dying neurons between the hippocampal subregions following a cortical contusion. In this study, we have used a model of controlled cortical impact and examined hippocampal pyramidal and granule neurons using FJB as a marker of neuronal death in order to test the following hypotheses: (1) the number of degenerating neurons correlates with injury severity, and (2) there are more CA3 pyramidal neurons lost after this type of insult.
Materials and methods Head trauma Young-adult male Sprague–Dawley rats (Harlan; 200– 225 g) were subjected to a mild or moderate unilateral cortical contusion (1 or 1.5 mm, respectively) as previously described (Scheff and Sullivan, 1999; Sullivan et al., 2002; Sullivan et al., 1998). The moderate cortical contusion used in these experiments results in severe behavioral deficits, significant loss of cortical tissue, blood–brain barrier disruption, and loss of hippocampal neurons (Baldwin and Scheff, 1996; Baldwin et al., 1997; Beer et al., 2000; Scheff et al., 1997, Sullivan et al., 1998), mimicking the sequelae of human closed-head injury. All subjects were anesthetized with isoflurane (2%) and placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA) prior to TBI. Using sterile procedures, the skin was retracted, and a point was identified midway between bregma and lambda and midway between the central suture and the temporalis muscle laterally. This location, which was consistent between all animals used in this study, constituted the central location of the 6 mm in diameter circular craniotomy that was made with a handheld Michele trephine (Miltex, York, PA). The skull cap was carefully removed without disruption of the underlying dura. Prior to the injury, the head of the animal was angled in a medial to lateral plane so that the impacting tip was perpendicular to the exposed cortical surface. This was accomplished by rotating the entire stereotaxic frame in the transverse plane while leaving the nose bar at 5.0. The exposed brain was injured using a pneumatically controlled impacting device (PSI, Fairfax, VA) with a 5-mm beveled tip, which compressed the
cortex at 3.5 m/s to a depth of either 1 or 1.5 mm (Baldwin and Scheff, 1996; Sullivan et al., 2000). Following injury, Surgicel (Johnson and Johnson, Arlington, TX) was laid upon the dura and the skull cap was replaced. A thin coat of dental acrylic was then spread over the craniotomy site and allowed to dry before the wound was stapled closed. During all surgical procedures and recovery, the core body temperature of the animals was maintained at 36–378C using heating pads. Animals were killed at 1 (1 mm, n = 4; 1.5 mm, n = 5), 2 (1 mm, n = 5; 1.5 mm, n = 5), or 7 (1 mm, n = 5; 1.5 mm, n = 5) days post injury for histological analysis. The Animal Care and Use Committee at the University of Kentucky approved all animal procedures. All efforts were made to minimize both the possible suffering and number of animals used. Tissue processing Rats were overdosed with sodium pentobarbital and transcardially perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde (PF) in PBS. The brains were removed from the cranium and postfixed in PF for 24 h at 48C before cryoprotecting in 20% sucrose/PBS at 48C. Brains were frozen in powdered dry ice and serial 50-Am cryosections through the entire extent of the hippocampal formation were cut on a sliding microtome in the coronal plane, placed into PBS, and mounted onto Fisher Superfrost Plus slides. Slides were dried on a slide warmer for 1 h and then processed for FJB and concurrent 4V,6-diamidino-2-phenylindole (DAPI) histochemistry. Histology and immunohistochemistry Staining with FJB and DAPI was performed using slight modifications of the technique described by Schmued and Hopkins (2000a). Sections were first incubated in a solution of 1% NaOH in 80% ethanol for 5 min and then were hydrated in graded ethanols (75, 50 and 25%; 5 min each) and distilled water. They were then incubated in a solution of 0.06% potassium permanganate for 10 min on a rotating stage, rinsed in distilled water for 2 min and incubated in a 0.0004% solution of FJB (HistoChem Inc., Jefferson, AR) and 0.0004% DAPI (Sigma, St. Louis, MO) for 20 min. Sections were then rinsed in distilled water (3 2 min), air-dried then placed on a slide warmer until fully dry (5–10 min). The dry slides were cleared in CitriSolv (2 5 min) and coverslipped with DPX (Fluka). Stereology The total number of FJB-positive neurons following TBI was determined blindly with respect to both severity and days post injury. Following sectioning of the entire
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extent of the hippocampal formation, a minimum of 12 equally spaced sections, with a variable starting location, were assessed employing an image analysis system (Bioquant Nova Prime v6.50.10, Nashville, TN) interfaced with an Olympus BX50 microscope and an Optronics VI470 camera (Goleta, CA). The entire reference volume (Vref) of the individual hippocampal subregions (dentate gyrus granule cells, CA3 pyramidal cells, and CA1 pyramidal cells) was estimated using the Cavalieri method (Michel and Cruz-Orive, 1988) and the anatomical boundaries as described by Amaral and Witter (1995). These various cell layers were identified using UV excitation of DAPI-positive neurons. Since the total number of FJB-positive neurons on any given section within each of the defined subregions often appeared to occur in clusters and didn’t appear to exceed more than 150 neurons, every FJB-positive neuron was counted under blue (450–490 nm) excitation light, employing a 20 objective to verify that the cell was a neuron. In this regard, individual small dissectors, often employed in unbiased stereological counting schemes (Gundersen et al.,
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1988) of large populations was not necessary to obtain an estimate of the individual section volume density. Estimates of the total number of FJB-positive neurons was P determined with the formula N v = [ (N / V subregion) / n] d Vref where N v = total estimated number of FBJ positive neurons in a subregion; N = total number of FJB-positive neurons counted in a subregion on an individual section; V subregion = volume of subregion on an individual section; n = number of individual sections analyzed; Vref = total reference volume for the subregion. Comparison of NeuroSilver and FJB staining An additional cohort of animals to those described above received a 1.5-mm contusion and were killed at 2 days post injury (n = 6). Alternate sections from these animals were processed for FJB and NeuroSilver histology (FD Neuro Technologies Inc., Baltimore, MD) and the total number of FJB-positive and NeuroSilver-positive neurons in the dentate gyrus were estimated using the same stereological techniques as described above.
Fig. 1. Fluoro-Jade B fluorescent neurons in the hippocampus following TBI. These are sections from animals that were sacrificed at 1 day post injury. (A) Area CA1 from an animal with a 1.5-mm injury; (B) Area CA1 from an animal with a 1-mm injury. (C) Area CA3 from an animal with a 1.5-mm injury; (D) Area CA3 from an animal with a 1-mm injury. (E) Dentate gyrus and hilus from an animal with a 1.5-mm injury; (F) Dentate gyrus and hilus from an animal with a 1-mm injury. Note the higher number of Fluoro-Jade positive neurons in all regions of animals that had received a 1.5-mm injury. Abbreviations: Pyr = Pyramidal cell layer; SG = Stratum granulosum of the dentate gyrus; Hi = Hilus of the dentate gyrus. Calibration bar = 100 Am.
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Data analysis The collected data were analyzed using a multi-factor analysis of variance with injury severity and days post injury used as main factors and subregions of the hippocampus used as a repeated measure within subject factor. For post hoc comparisons, the Tukey/Kramer test was used with significance set at P b 0.05. For comparison of FJBand NeuroSilver-stained neurons, a paired t test was used with significance also set at P b 0.05.
Results In all animals injured at both the mild and moderate level of TBI, there were obvious FJB-positive neurons in the hippocampus (Fig. 1). The statistical analysis revealed a significantly greater number of FJB-positive neurons [F = 17.059 df 1,46 P b 0.0004] in the hippocampal formation of moderately injured animals compared to the mild group. The analysis revealed a difference in days post injury [F = 4.210 df 2,46 P b 0.03] and as shown in Fig. 2, the greatest number of FJB-positive neurons was observed at 1 day post injury. The analysis revealed a significant regional distribution in FJB-positive neurons following the injury [F = 56.386 df 2,46 P b 0.0001]. A post hoc analysis revealed that the dentate gyrus granule cell layer had significantly more FJB-positive neurons than both the CA3 and CA1 regions and the CA3 region had significantly more positive neurons than CA1.
Fig. 2. Estimates of the total number of Fluoro-Jade B positive neurons in different regions of the hippocampus following either a mild or moderate cortical contusion. As early as 1 day following the injury, significant numbers of positive neurons were observed in the dentate gyrus granule cell layer. This number significantly increased with a moderate level of injury. The CA1 pyramidal cells showed the least sensitivity to the injury but did have significant number of positive cells with the moderate injury. The early time points post injury demonstrated the greatest numbers of FJBpositive cells. Bars represent means F SEM. Asterisks represent a significant difference from the 1-day post injury group as determined by Tukey/Kramer post hoc analysis ( P b 0.05).
Differences were further probed by analyzing changes in FBJ staining in mild and moderate injury separately. The analysis revealed no significant effect of days post injury for the mild injury severity [F = 0.580 df 2,22 P N 0.1] but did reveal a significant change in the regional distribution of FJB staining [F = 18.839 df 2,22 P b 0.0001] with the most significant differences between the dentate gyrus and hippocampal regions CA3 and CA1 as revealed by the post hoc analysis. CA3 did not differ significantly from CA1. Analysis of the moderately injured group demonstrated a significant days post injury effect [F = 4.307 df 2,24 P b 0.05] with post hoc analysis demonstrating significant differences between 1-day post injury and the 2- and 7-day post injury groups (Fig. 2). As with the mild injury group, there was a significant regional difference in the FJB staining in the moderate injured group [F = 46.635 df 2,24 P b 0.0001]. Post hoc analysis demonstrated that the dentate gyrus region had significantly more positive neurons than both CA3 and CA1 and that there were more positive CA3 neurons compared to area CA1. In order to compare FJB staining with NeuroSilver staining, we estimated the total number of positive neurons in the dentate gyrus at 2 days post injury (1.5 mm). There was no significant difference between the number of FJBpositive and NeuroSilver-positive neurons (FJB = 4146 F 443; NeuroSilver = 4746 F 916; P N 0.05) (Fig. 3).
Discussion In this study, we have demonstrated a regional difference in FJB staining among hippocampal subregions using a controlled cortical impact TBI model. Using both a mild and moderate level of injury, we demonstrated greater numbers of FJB-positive neurons as a function of increased injury severity. The dentate gyrus granule layer displayed the greatest number of injured neurons regardless of the level of severity with significant numbers of positive cells observed as early as 24 h following a mild injury. In addition, area CA3 showed significantly more FJB-positive neurons than area CA1 in the moderately injured group. This is the first quantitative report of extensive hippocampal granule cell injury following a cortical contusion model of TBI. Two previous reports employing a lateral fluid percussion (FP) model demonstrated FJB-positive granule cells but the number of cells was not quantified (Hallam et al., 2004; Sato et al., 2001). Similarly, two papers have reported on the time course and number of FJB-positive CA3 neurons following FP injury but did not estimate the total number of these neurons (Zhao et al., 2003; Zwienenberg et al., 2001). The present results coupled with the previously published findings support the idea that these hippocampal structures are sensitive to TBI. Previous studies have highlighted significant neuronal loss in the CA1 and CA3 regions of the hippocampus following a controlled cortical impact model (Baldwin et al., 1997; McCullers et al., 2002). A recent FP study has also
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Fig. 3. Photomicrographs of adjacent sections stained with NeuroSilver (A) and Fluoro-Jade B (B) in an animal subjected to a 1.5-mm injury and sacrificed 2 days post injury. Note the nearly identical pattern of staining in the stratum granulosum of the dentate gyrus (SG). Calibration bar = 100 Am.
demonstrated that, following trauma, the total number of hilar CA3 neurons as determined by stereology is decreased when examined 2 weeks after the injury (Grady et al., 2003). However, no significant differences were seen in the number of dentate gyrus granule neurons or hippocampal area CA1 pyramidal neurons as a function of injury. In our study, the largest number of FJB-positive neurons were seen in the granule cell layer of the dentate gyrus. However, estimates of the total number of granule cells range from 750,000 to 1,550,000 (Grady et al., 2003; Madeira et al., 1988; McCullers et al., 2002; Rapp and Gallagher, 1996; Rasmussen et al., 1996; West et al., 1991; Xavier et al., 1999). Thus, the proportion of neurons dying in the hippocampal dentate gyrus may be smaller than area CA3 (including the hilus) where the total number of neurons have been estimated to be between 267,600 and 400,000 (Grady et al., 2003; Rasmussen et al., 1996; West et al., 1991). Similarly, although few FJB-positive neurons were seen in area CA1, the total number of CA1 pyramidal neurons is similar to area CA3; estimated to be between 245,000 to 570,000 (Grady et al., 2003; McCullers et al., 2002; Rapp and Gallagher, 1996; Rasmussen et al., 1996; West et al., 1991; Xavier et al., 1999). Therefore, when
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described as a proportion of the total number of neurons, areas CA3 and CA1 may actually be impacted more than the granule cell layer of the dentate gyrus. While the total number of granule cells lost may be very small relative to the total number of neurons, this still may contribute to the behavioral and neurological deficits following TBI. As noted earlier, FJB appears to specifically stain degenerating neurons. We have also shown that FJB staining correlates very closely with more traditional markers of neuronal degeneration (NeuroSilver) in TBI. The advantage to using FJB lies in its ease of use, consistency, and that fact that is less capricious than silver methods. We have recently shown that while FJB stains dead or dying neurons following TBI, it does not label dying neurons in the spinal cord following a contusion injury (Anderson et al., 2003). The identity of the binding target of FJB is still unknown, although it is hypothesized that since FJB is a poly-anionic fluorescein derivative, it may label cationic products of degeneration such as spermidine, cadaverine, or putrescine (Schmued and Hopkins, 2000a). Although FJB does not appear to be a useful tool to study neuronal death in all models of neurotrauma, it does appear to be valuable in examining neuronal death in models of TBI (Anderson et al., 2003). FJB-positive neurons have been observed in the hippocampus following FP injury as early as 3 h after the insult with a peak at 1 day (Sato et al., 2001). These findings are similar to the findings of the current study where the peak number of neurons is generally seen at 1 day post injury. In addition, FJB-positive neurons appear to be greatly diminished by 7 days post injury following both FP (Sato et al., 2001) and cortical contusion (Fig. 2, present study). It is unclear at present if the 1-day post injury time point following a cortical contusion model of TBI represents the maximum FJB staining and thus it will be of interest to assess earlier time points post injury. Such an investigation may increase our understanding of the critical window for therapeutic intervention. In summary, FJB appears to give a bsnapshotQ of neurons that are dead or dying following cerebral injury and thus may be a reliable marker to test neuroprotective agents following TBI.
Acknowledgments This study was supported by the Kentucky Spinal Cord and Head Injury Research Trust #7–18, the Spinal Cord Research Foundation, and the NIH (NS 39828, AG21981). The authors gratefully acknowledge the technical assistance of Kelly Roberts.
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