It is known that various secondary biochemical events that occur following traumatic brain injury play an important role in aggravating the neuronal damage [1-3]. Brain which is severely damaged by an initial mechanical insult may be irreversibly damaged but secondary injury could be either prevented or ameliorated. One of the most important secondary biochemical events is the disruption of cellular calcium homeostasis. An excessive increase in intracellular Ca2+ via voltage- and receptor-gated Ca2+ channels following initial insult may activate Ca2+-dependent proteases and lipases, leading to eventual cell damage [1-3]. Calpain, a non-lysosomal cysteine protease, is activated by Ca2+ and breaks down cytoskeletal proteins such as microtubule-associated protein 2 (MAP2), which is abundant in perikarya and dendrites of neurons . In rats, a loss of MAP2 has been reported after traumatic brain injury [5-8] and degradation by calpain has been supposed to be the cause . It has also been reported that intracarotid infusion of calpain inhibitors improved neurological outcome  and reduced cytoskeletal proteolysis . In mice, a recent report showed that bolus i.v. administration of the calpain inhibitor SJA6017 improved neurological outcome without any ameriolation of alpha-spectrin breakdown .
The purpose of the present study was to investigate the effects of an i.v. infusion of calpain inhibitor 2 in a traumatic brain injury model of rats. We selected i.v. infusion as a regime, which is much simpler than intracarotid infusion in a clinical setting, assuming that calpain inhibitor 2 passes through the blood-brain barrier due to its highly lipophilic nature and low molecular weight. We compared its effects with those of moderate hypothermia, which is known to have neuroprotective potential [13,14].
The protocol of this study was reviewed and approved by the Ethics Committee for Animal Experimentation at Yamaguchi University School of Medicine.
Thirty-two male Wistar rats weighing 300-365 g and fasted for 12 h with unrestricted access to water were used. Anaesthesia was induced with isoflurane 4% in oxygen. After tracheal intubation, the lungs were mechanically ventilated and anaesthesia was maintained with isoflurane 1.5% in oxygen 33% and nitrogen. A polyethylene catheter (PE-50) was inserted via the tail artery for measurement of arterial pressure, blood gases, pH and glucose. Another PE-50 was inserted via the left femoral vein for drug administration. Rats were placed in a stereotaxic frame in the prone position. After infiltration with 0.25% bupivacaine, the skull was exposed by a midline incision and the scalp was reflected bilaterally. A craniotomy (diameter 8 mm) was made, carefully so as not to injure the dura, with a dental drill over the left parietal cortex with its centre 4 mm posterior to the bregma and 4 mm lateral to the midline. Right temporalis muscle temperature was monitored continuously and maintained at 37.5°C using a heating lamp.
After surgery, the rats were randomly assigned to four groups (n = 8, in each) as follows: calpain inhibitor treated (CI), hypothermic (HT), untreated (UT) and sham groups. Randomization was performed by drawing a ticket from an envelope. Traumatic brain injury was produced (see below) in the three groups CI, HT and UT. The CI group received calpain inhibitor 2 (Chemicon International Inc, CA, USA) diluted in normal saline (final concentration; 0.5 μmol mL−1) at a rate of 12 mL h−1 (0.2 mL min−1) i.v. for 5 min before injury. After injury, the infusion rate was reduced and maintained at 0.5 mL h−1 for 6 h (total volume: 4.0 mL; total dose: 2 μmol). Calpain inhibitor 2 is one of the peptide aldehyde agents, including calpain inhibitor 1, calpeptin, MDL28170 and SJA6017. These agents are known to have better transfer into neuronal cells than other calpain inhibitory agents, such as calpastatin. The rats in the HT group were cooled to 30°C (temporalis muscle temperature) by external cooling using an ice pack around the head and neck 45 min prior to injury. Rats were maintained at the target temperature for 60 min after injury and then warmed to 37.5°C for over 60 min. The UT group had injury with no treatment (received only vehicle infusion). The sham group received an identical surgical preparation without weight drop injury. In all animals, temporalis muscle temperature was maintained at 37.5°C except during the intended hypothermia period in the HT group.
Traumatic brain injury was produced by the weight drop method. A 5.6 g free falling weight was dropped through a cylinder of internal diameter 7.5 mm from a height of 30 cm onto an aluminum impactor tip (round, diameter 7.0 mm, 2.0 mm thick) placed over the intact dura in the area of the craniotomy. The stereotactic frame was rotated 30° laterally, so as to allow the weight drop to occur vertically onto the impactor tip. Following weight drop injury, the wound was closed, and the isoflurane was turned off except for the HT group where the isoflurane was maintained until the temperature of the temporalis muscle returned to 37.5°C after 60 min. After recovery from anaesthesia, the rats were extubated and kept in cages with free access to food and water.
At 6 h after weight drop injury or sham surgery, the rats were re-anaesthetized with 4% isoflurane. For histology or immunohistochemical analysis four rats from each group were perfusion fixed transcardially with 150 mL of warm saline followed by 4% 150 mL formaldehyde solution. The brain was removed and kept in 10% formalin in phosphate buffer fixative for 2 days at 4°C and then dehydrated and embedded in paraffin. Coronal sections of the brain including the primary injured cortical area (3.8-4.3 mm posterior to the bregma) were consecutively sectioned at a thickness of 6 μm. The avidin-biotin-peroxidase complex method (Vectastain ABC kits, Burlingame, CA, USA) was used for light microscopic analysis of MAP2. The antibody to rat brain MAP2 was purchased from Calbiochem (San Diego, CA, USA). Another adjacent section was stained with haematoxylin and eosin. Histological assessment was performed by an observer unaware of the treatment groups. Assessment for MAP2 staining in the dorsal hippocampus was undertaken using the five-point grading scale: 0, normal; 1, mild injury (over 75% of pyramidal cells in the dorsal hippocampus maintained); 2, moderate injury (50-74% maintained); 3, severe injury (25-49% maintained); 4, very severe injury (<25% maintained).
For western blotting analysis, four rats from each group were decapitated while anaesthetized and their brains removed and frozen immediately in liquid nitrogen. The brain was sagittally hemisected and fractionated to the cortex and hippocampus of the left hemisphere cut at the rostral and caudal edges adjacent to the primary injury area at −20°C. The fractionated cortex and hippocampus were then homogenized and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were performed according to the method previously reported . The antigen-antibody complex was visualized using the enhanced chemiluminescence western blotting detection kit. The MAP2 were quantified by an image analyzer (Densitograph AE-6905C; Atto, Tokyo, Japan). Antibody to rat brain MAP2 was the same as used for immunostaining. The standard was obtained from BioRad (Hercules, CA, USA), the western blotting detection kit from Amersham International (Buckinghamshire, UK). Protein concentration was measured according to the method of Bradford , using bovine serum albumen as a standard. The MAP2 concentration in each fraction was presented as a percentage assuming the sham control value as 100%.
The physiological data and western blotting were presented as mean ± standard deviation (SD) and were evaluated by ANOVA with Fisher's post hoc analysis. The grades of MAP2 staining were analysed by the non-parametric U-test. A value of P <0.05 was considered statistically significant.
All rats survived for 6 h after traumatic brain injury. Physiological variables are shown in Table 1. There were no significant differences in the pre-traumatic baseline values among the groups. Temporalis muscle temperature in the HT group decreased from 37.5 ± 0.1°C to 29.9 ± 0.1°C before trauma and was maintained at 30.0-30.1°C during the intended hypothermic period. Rectal temperature decreased to 32.2 ± 2.2°C. Partial pressure of arterial oxygen (PaO2) and plasma glucose values during the hypothermic period were significantly higher than the baseline values.
Haematoxylin and eosin staining.
In the injured groups, cortical defect and tissue contusion with intracerebral haemorrhage were seen in the left hemisphere (Fig. 1A,B). In the hippocampal region located beneath the cortical injury, the alignment of pyramidal cell layers showed disruption and neuronal structures were severely damaged with pyknotic change in the UT (Fig. 1C) and CI groups (Fig. 1D). In contrast, the structure of the pyramidal neurons in the HT group was better preserved, though not completely intact (Fig. 1E). No apparent neuronal damage was seen in both hemispheres in the sham group (Fig. 1F) and in the contralateral hemisphere in the three injured groups (not shown).
Representative microphotographs showing immunohistochemical reactions for MAP2 in the hippocampal CA1 region are shown in Figure 2. In all rats of both UT and CI groups, neuronal structures were severely deformed and the alignment of the pyramidal cell layer was disrupted and MAP2 immunostaining was decreased where the cell body disappeared (Figure 2A,B). In the HT group, alignment of the pyramidal cell layer and MAP2 immunostaining of neurons were fairly well maintained (Fig. 2C). In the sham group, no morphopathological derangement of neurons was observed and MAP2 immunostaining in the cell body and dendrites of the neurons appeared normal (Fig. 2D). The grades of MAP2 staining in the dorsal hippocampus are shown in Figure 3. In the sham group, all four rats showed Grade 0. In the three injured groups, the grades of MAP2 staining were significantly worse than those of the sham group (P < 0.05). In the HT group, the grades of MAP2 staining were significantly better than UT and CI groups (P < 0.05).
Western blotting analysis.
The results of immunoblotting for MAP2 and its quantified data 6 h after injury are shown in Figure 4. The mean MAP2 levels of the cortex tended to decrease in the UT (58 ± 44% of the sham control level), CI (59 ± 58%) and HT (68 ± 75%) groups, but the decrease did not reach statistical significance (Fig. 4a). There was no significant difference among the three injured groups. In contrast, the hippocampal MAP2 levels in the UT, CI and HT groups significantly decreased to 13 ± 9%, 28 ± 33%, 62 ± 25% of sham control level, respectively (Fig. 4b). The MAP2 level of the HT group was significantly higher than those of the UT and CI groups.
The present study demonstrated that traumatic brain injury induced by weight drop, which produced a severe irreversible cortical damage, produced a substantial decrease in MAP2 level in the ipsilateral hippocampus at 6 h after injury and that moderate hypothermia, but not calpain inhibitor 2, attenuated MAP2 degradation.
MAP2 is a principal protein component of microtubules which have many important functions such as mitotic movement of chromosomes, neurotransmitter release, structural growth and stabilization of neuritic processes . Thus, the damage to MAP2 may provoke various amounts of neuronal dysfunction.
It has been known that MAP2 is degraded by activation of calpain . Calpain, a Ca2+ dependent protease, is found throughout the nervous system and has two major isoforms (μ-calpain and m-calpain). Zhao and colleagues  demonstrated that μ-calpain increased gradually and showed a maximum at 6 h after controlled cortical impact injury, while the increase of m-calpain remained only approximately one quarter of that of μ-calpain. It is therefore suggested that a major part of calpain activation occurs during 6 h after injury. Although we did not measure both intracellular Ca2+ concentration and calpain activity in the present study, the possibility of activation of Ca2+ dependent calpain is quite likely.
The destruction of cytoskeletal proteins such as MAP2 and spectrin has been shown at early stage (10 min-3 h) in the experimental traumatic brain injury model [5-8]. It is possible that if calpain activation is blocked at an early stage after injury, subsequent cytoskeletal protein degradation may be prevented. The effects of calpain inhibition on cytoskeletal proteins have been studied at 24 h-7 days but not in the acute phase following traumatic brain injury. Also, it is suggested that a major part of calpain activation occurs during 6 h after injury. Therefore, the time of evaluation was selected at 6 h after trauma in the present study.
The brain injury model used in the present study is a modification of Feeney's brain contusion model produced by weight drop . The principal pathological changes were compression contusion to the cortex and neuronal damage in the ipsilateral hippocampus. The contused cortex was severely damaged by the primary injury and this assumed to be irreversible. We therefore focused on the neuronal MAP2 in the ipsilateral hippocampus located beneath the injured cortex, known to be vulnerable to ischaemia or hypoxia [20,21].
The neuroprotective effects of calpain inhibitors have been studied in traumatic brain injury. Saatman and colleagues  reported in the fluid percussion injury model in rats that a calpain inhibitor AK295, administered 15 min after injury via the carotid artery for 48 h, showed no significant improvement of neuromotor function at 48 h, but showed significant improvement of both neuromotor and cognitive functions at 7 days. However, in their successive study , they failed to observe any evidence that AK295 attenuates cortical lesion volume, regional apoptosis, or calpain-mediated spectrin proteolysis in vulnerable regions of the cortex and hippocampus. Posmantur and colleagues  demonstrated in rats that calpain inhibitor 2, administered via the carotid artery from 10 min to 24 h after cortical impact injury, significantly reduced the loss of neurofilament 68, neurofilament 200 and spectrin in the bilateral cortices. In the present study, we focused on whether continuous i.v. administration of calpain inhibitor 2 was effective in the traumatic brain injury model because i.v. administration is more clinically relevant than intracarotid administration. Calpain inhibitor 2, used in the present study, is highly lipophilic, has a relatively low molecular weight (401.6) and thus may be easily permeable to the blood-brain barrier. In the rat subarachnoid haemorrhage model, Germano and colleagues  reported the neuroprotective effects of calpain inhibitor 2 administered i.v. Penetration of this drug could have been facilitated by the injury-induced disruption of the blood-brain barrier. Nevertheless, we failed to show an ameriolating effect despite the treatment being started before insult. The total dose, 2.0 μmol given i.v. over a 6 h period, is approximately three times the dose used during the 6 h period in Posmantur's study . The dose used in the present study is also greater than that of a less potent calpain inhibitor 1 (0.4 μmol mL−1, 1.0 mL) which significantly suppressed fodrin (spectrin) degradation in an ischaemia-reperfusion model  and that of a successful dose in a subarachnoid haemorrhage model . The results suggest that calpain inhibitor 2 with the tested regime does not significantly attenuate MAP2 degradation in the ipsilateral hippocampus after traumatic brain injury. There may be a possibility that some proteolytic enzymes other than calpain might be involved in MAP2 degradation and neuronal damage.
Kupina and colleagues  recently reported in a mouse model of diffuse brain injury that bolus i.v. administration of calpain inhibitor SJA6017 improved neurological outcome without any ameriolation of alpha-spectrin breakdown. SJA6017 is peptide aldehyde agent known to have a similar effect to calpain inhibitor 1 and 2. Therefore, we have no clear explanation for the contrary results. Due to the small sample size in the experimental groups, the possibility that calpain inhibitor 2 may have some neuroprotective effect cannot be excluded. However, there was a significant difference between the CI and HT groups for grades of MAP2 immunostaining and MAP2 content, these being significantly better maintained in the HT group. Therefore, the neuroprotective effect of calpain inhibitor 2 with the tested regime, if any, is significantly less than that of hypothermia.
The neuroprotective effects of hypothermia in brain injury in human beings are still a controversial issue [24,25] but have been consistently demonstrated in experimental brain injury models [13,14]. In the present study, we demonstrated in the hippocampus that the degradation of MAP2 induced by weight drop injury was significantly attenuated by moderate hypothermia (30°C). Our findings are similar to those of Taft and colleagues , who demonstrated protective effects of hypothermia against hippocampal MAP2 degradation in a fluid percussion injury model. Besides suppressing glutamate release and intracellular calcium influx, hypothermia has many other favourable effects such as reducing free radical production and blood-brain barrier disruption as well as producing metabolic suppression [26-28]. It remains unknown whether a longer duration (approximately 50 min) of anaesthesia with isoflurane in the hypothermic group have partly contributed to preservation of MAP2 in the present study.
In conclusion, moderate hypothermia (30°C), but not calpain inhibitor 2 with the tested regime, attenuated the proteolysis of hippocampal MAP2. Failure of calpain inhibitor 2 to attenuate neuronal damage indicates the involvement of multifactorial mechanisms in the pathophysiology of traumatic brain injury.
Supported in part by the Ministry of Education, Science, Sports and Culture Grant No. 11770852.
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