Material and methods lower rod end had an impactor tip attached i.e., that part

H .Q. Yan et al. Brain Research 887 2000 134 –143 135 be better clarified and enhanced while minimizing potential 4 isoflurane Anaquest, Memphis, TN in oxygen and the nonspecific effects, which can reduce therapeutic efficacy. rats intubated with a 14-gauge angiocatheter and me- Consequently, the best protocol and patient populations for chanically ventilated with 2.0 isoflurane 66 N O bal- 2 the use of hypothermia in clinical TBI remain to be defined ance O for surgery. Venous and arterial catheters were 2 and optimized. One approach that may prove useful is to placed in the tail for FGF-2 infusion and the continuous combine other treatments with hypothermia that are in- monitoring of blood pressure and arterial glucose and dependently effective and have other beneficial specific blood gas sampling. A rectal probe was inserted for mechanistic actions. The use of exogenous trophic factors continuous monitoring of body temperature, and a micro- is one class of candidates, especially since there is probe thermistor Type MT-29 1, Physiotemp Instruments evidence that TBI alters neurotrophin receptors after injury Inc., Clifton, NJ was inserted into a temporalis muscle for [39] and hypothermia treatment can reduce the endogenous indirect monitoring of brain temperature. A midline inci- neurotrophic response of the brain after experimental CCI sion was made, the soft tissues reflected, and a 7 mm [11,23,24]. craniotomy was made between lambda and bregma and A number of studies in experimental TBI have shown centered 5 mm laterally on the right side of the central that neurotrophins attenuate functional deficits or his- suture. The rats received a CCI through the craniotomy at a topathology [16,44–46]. One of several neurotrophic fac- velocity of 4 m s with a tissue deformation depth of 2.6 tors, fibroblast growth factor-2 FGF-2 has been shown mm. Shams underwent identical surgical procedures, but protective in a number of in vitro injury models [33,37,52], did not receive the impact. After injury, the scalp incision and in vivo models such as fluid percussion TBI was closed with interrupted nylon sutures and the rat was [12,25,38], stroke [27,28], and spinal cord injury [31,40]. removed from the injury device and returned to the heating Importantly, FGF-2 is effective when administered after pad. The rat was stabilized on anesthesia for 30 min after injury, and retards functional recovery when its endogen- which arterial blood gas ABG, glucose, and hematocrit ous expression is inhibited [42]. were checked just before injury. Mean arterial pressure Given the previous beneficial effects of FGF-2, we was kept above 90 mm Hg, brain and body temperature combined FGF-2 treatment with moderate hypothermia were kept at 3760.58C, and pCO at 4065 mm Hg. 2 therapy to determine if synergistic effects occur by evaluating the effect of individual or combined FGF-2 and 2.3. Injury device moderate hypothermia therapy on motor, cognitive, and histological outcome following a moderate CCI injury in The CCI injury device [15] consisted of a small 1.975 rats. cm bore, double-acting, stroke-constrained, pneumatic cylinder with a 5.0 cm stroke. The cylinder was rigidly mounted in an angled vertical position on a crossbar. The

2. Material and methods lower rod end had an impactor tip attached i.e., that part

of the shaft that comes into contact with the exposed dura 2.1. Subjects and the upper rod end was attached to the transducer core of a linear velocity displacement transducer LVDT. The Fifty adult male Sprague–Dawley rats weighing 370– velocity of the impactor shaft was controlled by gas 400 g on the day of surgery were used. Animals were pressure and measured directly by the LVDT Shaevitz group-housed two per cage in standard steel wire mesh Model 500 HR; Macro Sensors, Pennsauken, NJ, which cages with ad libitum access to food and water. All produced an analog signal that was recorded by a PC- procedures carefully conformed to the guidelines outlined based data acquisition system Axoscope; Axon Instru- in the Guide for the Care and Use of Laboratory Animals ments, Inc., Foster City, CA for analysis of time displace- from the U.S. Department of Health and Human Services ment parameters of the impact. and were approved by the University of Pittsburgh Institu- tional Animal Care and Use Committee. The animals were 2.4. FGF-2 infusion and hypothermia induction randomized to one sham or four CCI groups: Sham1 vehicle VEH; FGF-2 45 mg kg h for 3 h i.v.1 Ten minutes after injury, rats randomized to the hypo- Normothermia 3760.58C; FGF-2 same as above1 thermic groups were cooled to a brain temperature of hypothermia 3260.58C for 3 h; VEH1Norm; VEH1 3260.58C with ice packs; those randomized to normother- Hypo. mic groups were maintained at 3760.58C with a heating pad. Hypothermic rats were maintained at 3260.58C for 3 2.2. Surgical and injury protocols h, and then gradually over a 1 h period rewarmed to 3760.58C. Normothermic rats were maintained at Prior to surgery, previously trained animals were tested 3760.58C for 4 h, beginning 10 min post injury, as were on both the beam balance and beam walking tasks to the sham controls. Infusion of FGF-2 began 30 min after establish a baseline measure. Anesthesia was induced with injury for 3 h at a dose of 45 mg kg h for a total dose of 136 H 135 mg kg of FGF-2. The sham rats were infused with before being placed in a heated incubator between trials 4 vehicle at the same rate and for the same time as the CCI min intertrial interval. groups. All lines were subsequently removed at 4 h, anesthesia was discontinued, and the animals were allowed 2.6. Tissue preparation to regain consciousness before being returned to their home cages. Four weeks after injury, animals were deeply anes- thetized with pentobarbital Nembutal, 80–100 mg kg; 2.5. Functional assessments Abbott Laboratories, North Chicago, IL and transcardially perfused with 100 ml of 0.1 M phosphate buffered saline 2.5.1. Motor performance with 5 U ml heparin pH 7.4, followed by 500 ml 4 Gross vestibulomotor function was assessed on a beam- paraformaldehyde. After perfusion, the brain was removed balance task that consisted of placing the animal on a and immersed in 4 paraformaldehyde in 0.1 M phosphate suspended, narrow wooden beam 1.5 cm wide and buffer pH 7.4 at 48C overnight. The brain was next recording the duration it remained on the beam for a transferred to 15 sucrose in 0.1 M phosphate buffer pH maximum of 60 s. Training prior to injury consisted of 7.4 at 48C for 24 h then to 30 sucrose in 0.1 M three trials, during which baseline measurements were phosphate buffer pH 7.4 at 48C until sunken. The cryop- taken. More complex vestibulomotor function and coordi- rotected rat brain was frozen and two series of 7 mm thick nation were assessed using a modified beam-walking task. coronal sections were cut on a cryostat Jung CM 1800; Briefly, this task consisted of training rats to escape a Brodersen Instruments, Valencia, PA and mounted on bright light and high decibel white noise Lafayette gelatinized glass slides. One set of sections was stained Instruments, Inc., Layfaette, IN, Model [15800C by with Cresyl violet for morphological analysis and the other traversing a narrow wooden beam 2.53100 cm and set was prepared for immunohistochemistry. entering a darkened goal box at the opposite end. Noxious stimuli were terminated when the rat entered the goal box. 2.6.1. Morphology Four pegs 3.0 mm diameter, 4.0 cm high were placed in Treatment efficacy on the survival of vulnerable neurons an alternating sequence along the beam to increase the in the hippocampal CA and CA regions was evaluated 1 3 difficulty of the task. Performance was assessed by the from coronal sections underlying the area of contusion, latency to traverse the beam. Data for each session approximately 3.5 mm posterior to bregma, using a double consisted of the mean of three trials. All rats were pre- blind analysis for each of the following groups Sham1 trained prior to injury. VEH56; FGF-21Norm54; VEH1Norm54; FGF-21 Hypo54, and VEH1Hypo55. To reduce counting errors 2.5.2. Cognitive performance associated with false positive identification of dying neu- A Morris water maze task variant was used to compare rons, the total number of CA and CA surviving neurons 1 3 acquisition rates between groups. The maze consisted of a were counted using a Nikon Eclipse E600 microscope plastic pool 180 cm in diameter and 60 cm in depth filled Nikon Corporation, Tokyo, Japan. All data are reported with water to a depth of 28 cm with a clear Plexiglas stand as the percent of total percent neurons in the ipsilateral 10 cm in diameter and 26 cm high, i.e., 2 cm below the injured CA and CA regions relative to the contralateral 1 3 water’s surface used as the hidden goal platform. The pool side. Cortical volume was determined by outlining both the was located in a 2.532.5 m room with numerous extra- contralateral and ipsilateral injured hemispheres MCID, maze cues e.g., posters that remained constant throughout Imaging Research, Ontario, Canada then subtracting the the experiment. Water maze testing began on day 14 area of the ipsilateral hemisphere from the contralateral post-injury in order to avoid possible confounds with the and summing the differences to calculate a mean volume. motor deficits observed in the first few days following injury. The rats were given four trials per day for five 2.6.2. Immunohistochemistry consecutive days to assess performance. For each daily Glial fibrillary acidic protein GFAP immunohisto- block of four trials, the subjects were placed in the pool chemistry was conducted on paraffin-embedded tissue. facing the wall. Trials were initiated from each of the four Sections were pre-blocked with 10 normal rabbit serum possible start locations north, east, south, west in a NRS and 0.1 Triton X-100 in 0.1 M PBS TX-PBS. randomized manner. The goal platform was positioned 45 Sections were incubated with primary antibody, mouse cm from the outside wall and was placed in the north east, anti-GFAP monoclonal antibody 1:3000; Chemicon Inter- south east, south west, or north west quadrant of the maze. national, Temecula, CA with 5 NRS and TX-PBS at 48C The location of the platform was held constant for each for 16–24 h. Affinity-purified rabbit anti-mouse IgG 1:50; animal. A maximum of 120 s was allowed to each rat to Jackson ImmunoResearch Laboratories, West Grove, PA find the hidden platform. If the rat failed to find the was incubated as secondary antibodies with 5 NRS and platform within the allotted time, it was placed on the TX-PBS at 48C for 2 h on a shaker. Mouse peroxidase– platform by the experimenter where it remained for 30 s anti-peroxidase PAP soluble immune complexes 20 g H .Q. Yan et al. Brain Research 887 2000 134 –143 137 ml; Jackson ImmunoResearch Laboratories, West Grove, 3. Results PA were used to visualize immunoreactivity with 2 NRS and TX-PBS at 48C for 3 h. Tissue slides were rinsed 3.1. Physiology between all steps with TX-PBS three times for at least 10 min each time. The peroxidase reaction was developed Arterial blood gases were measured at 10, 30, 60, 120, with DAB Substrate Kit Vector; Burlingame, CA until a 180 and 240 min post injury Table 1. Mean arterial blood dark brown reaction product was evident. Sections were pressure MABP was never below 90 mmHg in any rat rinsed in water, dehydrated in alcohols, defatted in although some slight group differences were seen Fig. 1. xylenes, and coverslipped for light microscopic analysis. As seen in Table 1, slight elevations in blood glucose Some sections were counterstained with hematoxylin levels were observed during hypothermia before returning Vector; Burlingame, CA. At least three sections through near baseline after rewarming 240 min after TBI in both the hippocampus of each animal were processed for hypothermic groups. Hypothermia is known to increase immunostaining. Control experiments were run in parallel circulating catecholamines [26] which is likely responsible to confirm specificity. Primary antibody was omitted and for the slight increase in glucose seen in our hypothermic polyclonal antibody to GFAP DAKO, Denmark was used rats. for comparison. 3.2. Motor performance 2.7. Statistical analysis A repeated-measures ANOVA on beam balancing ability revealed a significant group difference F 54.066, P5 4,45 Analyses were performed using Statview software on a 0.0067, a significant time days difference F 5 5,225 Macintosh computer. Behavioral data were analyzed using 28.397, P,0.0001, and a significant group3day inter- repeated-measures analysis of variance ANOVA. If a action F 53.614, P,0.0001. Post-hoc analysis re- 20,225 significant effect was found with the ANOVA, individual vealed that all CCI rats had motor deficits compared to the group comparisons were made with Fisher’s PLSD post- Sham1VEH group P,0.001. However, there were no hoc tests. Histological and physiological data were ana- significant differences among the injured groups Fig. 2. lyzed with unpaired t-tests. Data were expressed as In contrast, a repeated measures ANOVA of the beam mean6S.E.M. and a significance level of P,0.05 was walking task also showed a significant group F 5 4,45 used. 17.181, P,0.0001, time F 586.633, P,0.0001, and 5,225 Table 1 Systemic physiological parameters Baseline VEH1Sham VEH1Norm FGF-21Norm VEH1Hypo FGF-21Hypo pH 7.4260.01 7.4360.01 7.4360.01 7.4460.01 7.4460.01 pCO 42.760.7 41.461.0 40.961.0 40.660.7 40.760.9 2 pO 169.066.9 171.363.4 174.662.2 175.066.6 177.863.4 2 p. Glu mg 155.664.6 151.064.3 141.862.7 160.666.1 147.967.2 30 min pH 7.4460.01 7.4460.01 7.4560.01 7.4660.01 7.4660.00 pCO 38.860.5 38.960.7 36.460.7 37.360.6 36.160.3 2 pO 174.265.1 178.764.3 181.664.5 201.163.5 198.264.1 2 p. Glu mg 160.465.9 163.466.6 158.365.2 214.2612.2 191.9610.3 120 min pH 7.4460.01 7.4260.01 7.4260.01 7.4460.01 7.4460.01 pCO 38.760.5 38.461.1 38.960.8 37.960.5 37.360.8 2 pO 179.563.5 175.063.5 176.465.3 198.064.5 197.563.0 2 p. Glu mg 159.766.2 151.667.1 153.464.6 209.3616.5 193.1615.1 240 min pH 7.4260.01 7.4160.01 7.4260.01 7.4060.02 7.3960.01 pCO 39.760.7 39.060.8 38.060.9 40.260.8 40.561.0 2 pO 176.764.6 175.064.2 175.666.5 175.764.4 170.865.5 2 p. Glu mg 160.264.2 148.867.4 154.763.8 200.1621.3 163.5610.3 Both hypothermia groups are significant over time, as well as over all other groups P,0.05. Data at 10, 60, and 180 min not shown, but are consistent with other intra group findings. p. Glu5plasma glucose. 138 H Fig. 1. Mean arterial blood pressure MABP data before and 4 h after the injury in all the animal groups. Values are mean 6S.E.M.. group3day interaction F 56.488, P,0.001. Post- 3.3. Cognitive performance 20,225 hoc analysis showed that the combined FGF-21hypo group displayed significantly improved beam walking A repeated measures ANOVA revealed significant dif- function compared to the other groups P,0.0042 vs. ferences in the latency to find a hidden platform among FGF-21Norm, P,0.0021 vs. VEH1Norm, and P,0.0153 groups F 59.42, P,0.0001 and time F 537.869, 4,45 4,180 vs. VEH1Hypo. P,0.0001, but there was no significant group3day Fig. 2. Mean 6S.E.M. beam walk latency scores prior to, and Days 1–5 following, CCI injury. Injured animals treated with 135 mg kg of FGF-2 or 3 h hypothermia after injury did not differ significantly from the control injury group. The FGF-2 combined with hypothermia treatment had a significantly better outcome over time than either treatment alone. H .Q. Yan et al. Brain Research 887 2000 134 –143 139 interaction F 50.609, P50.874. Post-hoc analyses groups. The percentage of CA neuronal survival was 16,180 1 showed that all injured groups had deficits P,0.001. 98.4965.68 Sham1VEH, 35.4364.13 FGF-21Norm, Additionally, all treated but injured groups showed re- 34.9467.69 VEH1Norm, 35.4364.13 FGF-21Hypo, duced deficits compared with untreated injured animals and 43.6269.71 VEH1Hypo. All injured groups were P,0.05 however, there were no differences seen be- significantly different from the Sham1VEH group P, tween treatment groups Fig. 3. Post-hoc analysis showed 0.05, but no significant differences were observed among significant differences at days 14, 15, and 18 with both the injured P.0.05, unpaired t-test. The mean percentage of FGF-21Norm and VEH1Hypo groups performing better CA neuronal survival was 104.8866.22 Sham1VEH, 3 than the VEH1Norm group P,0.05. The FGF-21Hypo 54.2967.45 FGF-21Norm, 53.5166.75 VEH1Norm, group exhibited a significantly reduced latency to find the 73.3268.92 FGF-21Hypo, and 76.7569.67 VEH1 hidden platform compared to the VEH1Norm group only Hypo. An unpaired t-test on cortical tissue loss did not at post-operative day 18 P,0.029, Fisher. In addition, all reveal a significant difference among injured groups P. injured groups with or without treatment demonstrated 0.05, n.s.. The mean 6S.E.M. cortical volume was 3 3 significantly shorter swim latencies P,0.05 during the 22.3463.07 mm FGF-21Norm, 23.4163.30 mm 3 visible platform trials suggesting that their performance VEH1Norm, 21.1063.23 mm FGF-21Hypo, and 3 deficits during the hidden platform trials were not con- 20.4761.62 mm VEH1Hypo. founded by non-specific deficits such as visual processing and motivation. 3.4.2. Immunohistochemistry 3.4. Histology GFAP staining showed marked astrogliosis in all injured brains Fig. 4. An increase in GFAP-immunoreactivity 3.4.1. Neuronal density was observed in the ipsilateral hippocampus cortex and Hippocampal CA and CA neuronal density underlying thalamus in all injured groups Fig. 4. GFAP staining in 1 3 the contusion was examined from each group. TBI pro- the ipsilateral CA corresponded directly to the area of 3 duced significant reductions in CA and CA surviving maximal hippocampal cell loss caused by CCI injury. 1 3 neurons in the hippocampus ipsilateral to impact compared However, FGF-2 and or hypothermia-treated animals to the contralateral hippocampus in all the CCI-injured showed an attenuation of GFAP expression compared to Fig. 3. The effect of post-CCI FGF-2 and hypothermia treatment on Morris water maze place learning after TBI. Graph plotting the latencies to find a hidden platform beginning 2 weeks after TBI. Injured animals treated with 135 mg kg of FGF-2 and or 3 h hypothermia after injury had significantly shorter latencies to find the hidden platform than the VEH1Norm group. However, the combination was not better than FGF-2 or hypothermia alone. 140 H Fig. 4. Photomicrograph of GFAP-immunoreactivity counterstained with hematoxylin at 4 weeks after CCI injury revealing marked astrogliosis in all injured brains D–O compared to the sham controls A–C. An attenuation of GFAP over-expression is observed in the VEH1Hypo G–I, FGF-21Norm J–L, and FGF-21Hypo M–O groups compared to the VEH1Norm D–F group. Scale bar 2 mm A,D,G,J,M, 500 mm B, E, H, K, N, 100 mm C, F, I, L, O. H .Q. Yan et al. Brain Research 887 2000 134 –143 141 the injured VEH1Norm-treated control by immunohisto- produces less severe histological damage. These data chemistry Fig. 4. showed that the histopathological consequences of these different types of injury are quite different. It may be that FGF-2 histological protection is limited by injury intensity,

4. Discussion but that functional benefit can still be obtained by modulat-