The Role of Acid Sphingomyelinase Inhibition in Repetitive Mild Traumatic Brain Injury.

BACKGROUND: Chronic traumatic encephalopathy is a consequence of repetitive mild traumatic brain injury (rmTBI). These injuries can result in psychiatric disorders that are treated with amitriptyline. Amitriptyline improves neuronal regeneration in major depression via inhibition of acid sphingomyelinase. We hypothesized that acid sphingomyelinase inhibition would preserve neuronal regeneration and decrease depressive symptoms following rmTBI in a murine model. METHODS: A murine model of rmTBI was established using a weight-drop method. Mice were subjected to mTBI every other day for 7 d. Mice received amitriptyline injection 2 h prior to each mTBI. After the final mTBI, mice underwent behavioral studies or biochemical analysis. Hippocampi were analyzed for markers of neurogenesis and phosphorylated tau aggregation. RESULTS: Mice that underwent rmTBI showed increased hippocampal phosphorylated tau aggregation 1 mo following rmTBI as well as decreased neuronal regeneration by bromodeoxyuridine uptake and doublecortin immunohistochemistry. Mice with either genetic deficiency or pharmacologic inhibition of acid sphingomyelinase demonstrated improved neuronal regeneration and decreased phosphorylated tau aggregation compared to untreated rmTBI mice. Behavioral testing showed rmTBI mice spent significantly more time in the dark and waiting to initiate feeding compared to sham mice. These behaviors were partially prevented by the inhibition of acid sphingomyelinase. CONCLUSIONS: We established a murine model of rmTBI that leads to tauopathy, depression, and impaired hippocampal neurogenesis. Inhibition of acid sphingomyelinase prevented the harmful neurologic and behavioral effects of rmTBI. These findings highlight an important opportunity to improve recovery or prevent neuropsychiatric decline in patients at risk for chronic traumatic encephalopathy.

Role of Akt and mammalian target of rapamycin in functional outcome after concussive brain injury in mice.

Akt (protein kinase B) and mammalian target of rapamycin (mTOR) have been implicated in the pathogenesis of cell death and cognitive outcome after cerebral contusion in mice; however, a role for Akt/mTOR in concussive brain injury has not been well characterized. In a mouse closed head injury (CHI) concussion traumatic brain injury (TBI) model, phosphorylation of Akt (p-Akt), mTOR (p-mTOR), and S6RP (p-S6RP) was increased by 24 hours in cortical and hippocampal brain homogenates (P<0.05 versus sham for each), and p-S6RP was robustly induced in IBA-1+ microglia and glial fibrillary acidic protein-positive (GFAP+) astrocytes. Pretreatment with inhibitors of Akt or mTOR individually by the intracerebroventricular route reduced phosphorylation of their respective direct substrates FOXO1 (P<0.05) or S6RP (P<0.05) after CHI, confirming the activity of inhibitors. Rapamycin pretreatment significantly worsened hidden platform (P<0.01) and probe trial (P<0.05) performance in CHI mice. Intracerebroventricular administration of necrostatin-1 (Nec-1) before CHI increased hippocampal Akt and S6RP phosphorylation and improved place learning (probe trials, P<0.001 versus vehicle), whereas co-administration of rapamycin or Akt inhibitor with Nec-1 eliminated improved probe trial performance. These data suggest a beneficial role for Akt/mTOR signaling after concussion TBI independent of cell death that may contribute to improved outcome by Nec-1.

Significance of ubiquitin carboxy-terminal hydrolase L1 elevations in athletes after sub-concussive head hits.

The impact of sub-concussive head hits (sub-CHIs) has been recently investigated in American football players, a population at risk for varying degrees of post-traumatic sequelae. Results show how sub-CHIs in athletes translate in serum as the appearance of reporters of blood-brain barrier disruption (BBBD), how the number and severity of sub-CHIs correlate with elevations of putative markers of brain injury is unknown. Serum brain injury markers such as UCH-L1 depend on BBBD. We investigated the effects of sub-CHIs in collegiate football players on markers of BBBD, markers of cerebrospinal fluid leakage (serum beta 2-transferrin) and markers of brain damage. Emergency room patients admitted for a clinically-diagnosed mild traumatic brain injury (mTBI) were used as positive controls. Healthy volunteers were used as negative controls. Specifically this study was designed to determine the use of UCH-L1 as an aid in the diagnosis of sub-concussive head injury in athletes. The extent and intensity of head impacts and serum values of S100B, UCH-L1, and beta-2 transferrin were measured pre- and post-game from 15 college football players who did not experience a concussion after a game. S100B was elevated in players experiencing the most sub-CHIs; UCH-L1 levels were also elevated but did not correlate with S100B or sub-CHIs. Beta-2 transferrin levels remained unchanged. No correlation between UCH-L1 levels and mTBI were measured in patients. Low levels of S100B were able to rule out mTBI and high S100B levels correlated with TBI severity. UCH-L1 did not display any interpretable change in football players or in individuals with mild TBI. The significance of UCH-L1 changes in sub-concussions or mTBI needs to be further elucidated.

Distinct time courses of secondary brain damage in the hippocampus following brain concussion and contusion in rats.

Secondary brain damage (SBD) is caused by apoptosis after traumatic brain injury that is classified into concussion and contusion. Brain concussion is temporary unconsciousness or confusion caused by a blow on the head without pathological changes, and contusion is a brain injury with hemorrhage and broad extravasations. In this study, we investigated the time-dependent changes of apoptosis in hippocampus after brain concussion and contusion using rat models. We generated the concussion by dropping a plumb on the dura from a height of 3.5 cm and the contusion by cauterizing the cerebral cortex. SBD was evaluated in the hippocampus by histopathological analyses and measuring caspase-3 activity that induces apoptotic neuronal cell death. The frequency of abnormal neuronal cells with vacuolation or nuclear condensation, or those with DNA fragmentation was remarkably increased at 1 hr after concussion (about 30% for each abnormality) from the pre-injury level (0%) and reached the highest level (about 50% for each) by 48 hrs, whereas the frequency of abnormal neuronal cells was increased at 1 hr after contusion (about 10%) and reached the highest level (about 40%) by 48 hrs. In parallel, caspase-3 activity was increased sevenfold in the hippocampus at 1 hr after concussion and returned to the pre-injury level by 48 hrs, whereas after contusion, caspase-3 activity was continuously increased to the highest level at 48 hrs (fivefold). Thus, anti-apoptotic-cell-death treatment to prevent SBD must be performed by 1 hr after concussion and at latest by 48 hrs after contusion.

[Computer analysis of erythrocyte catalase activity in diagnosis of mild brain trauma and concussion].

A complex investigation of catalase activity in erythrocytes was conducted basing on the results of computer quantitative morphometry of histochemical examination of blood samples from persons with mild craniocerebral trauma. It was found that erythrocytic catalase activity correlates with severity of brain trauma. A novel technique of objective histochemical diagnosis of trauma is described. It provides objective grounds for expert conclusions. Differential features of erythrocytic shape were defined in a small series of micropreparations vs the rest blood samples.

[Significance of histochemical parameters of erythrocyte catalase activity for diagnosing mild concussion and contusion of the brain]. / Znachenie gistokhimicheskikh pokazatelei katalaznoi aktivnosti éritrotsitov dlia diagnostiki sotriaseniia i ushiba golovnogo mozga legkoé stepeni.

A total of 227 primary and repeated forensic medical expert conclusions in victims with slight craniocerebral injuries (CCI) are analyzed. Erythrocyte catalase (CTer) activity was analyzed on the basis of the results of computer quantitative morphometry of histochemical analysis of blood smears from 50 victims with slight CCI. CTer activity correlated with the severity of CCI. A new objective method for histochemical diagnosis of slight concussion and contusion of the brain is suggested, which rules out errors in forensic medical expert evaluations in examinations of this category of victims.

Nitric oxide synthase expression after human brain contusion.

OBJECTIVE: Nitric oxide is a universal mediator of biological effects in the brain, and it has been implicated in the pathophysiological processes of traumatic brain injury. Experimental studies have indicated posttraumatic up-regulation of the three different isoforms of nitric oxide synthase (NOS) (i.e., inducible NOS [iNOS], endothelial NOS, and neuronal NOS) after brain trauma. This study was undertaken to investigate the cellular sources and tissue compartments of nitric oxide produced by human patients undergoing surgical treatment for contusional brain injuries. METHODS: Contused brain tissue specimens from eight consecutive patients who underwent surgical treatment for brain contusions 3 hours to 5 days after trauma were evaluated in immunohistochemical analyses. Double-staining assays were used to define which cells produced the different isoforms. RESULTS: Increases in iNOS-positive cells were detectable within 6 hours after trauma, with a peak at 8 to 23 hours. Expression of iNOS after trauma was detected in neurons, macrophages, neutrophils, astrocytes, and oligodendrocytes. The cellular sources of iNOS differed at different times after trauma. No detectable difference in the expression of the neuronal or endothelial isoforms was observed for trauma patients, compared with control subjects. CONCLUSION: iNOS expression was up-regulated in a time-dependent manner in human brain tissue obtained from patients undergoing surgical treatment for contusional trauma. Our human data largely parallel experimental findings in rats, indicating that such trauma models are relevant for experimental studies and treatment trials.

Effects of deferoxamine on tissue superoxide dismutase and glutathione peroxidase levels in experimental head trauma.

BACKGROUND: This study aims to evaluate the effects of deferoxamine on tissue superoxide dismutase (SOD) and glutathione peroxidase (GPx) brain levels after head trauma. METHODS: Thirty rabbits were divided equally into three groups: group 1 was the sham-operated group, group 2 suffered head trauma (no treatment was given), and group 3 received deferoxamine 50 mg/kg after the trauma. Head trauma was applied unilaterally. One hour after trauma, brain cortices were resected and SOD and GPx levels were determined. One-way analysis of variance and Tukey-HSD tests were used for analysis. Significance was defined as p < 0.05. RESULTS: Baseline SOD levels are preserved in the traumatized side of the deferoxamine-treated group. Although GPx level of the traumatized side of the deferoxamine-treated group decreased significantly, the decrease was significantly less than the nontreated group. CONCLUSION: Trauma leads to a decrease in brain tissue SOD and GPx levels. Deferoxamine suppresses this decrease completely in SOD level and partially in GPx level when given after trauma.

Time profile of neuron specific enolase serum levels after experimental brain injury in rat.

The aim of this study was to investigate the time course of NSE serum levels after traumatic brain injury in rats. 65 male Wistar rats were subjected to severe cortical impact injury (100 PSI, 2 mm deformation). Blood samples were drawn directly after trauma, after 1 h, 6 h, 12 h, 24 h, and 48 h in the trauma group as well as in sham operated animals directly after craniotomy, after 6 h and after 48 h. NSE serum levels were estimated with a commercially available enzyme immuno assay (LIA-mat Sangtec). The control animals showed a NSE serum level of 8.82 micrograms/l (mean, n = 10). We demonstrated a time dependent release of NSE into the serum after trauma. The highest NSE serum values were detected six hours after trauma (31.5 micrograms/l, mean, n = 10). NSE serum level seems to reflect neuronal damage after cortical contusion in the rat in a time dependent manner.

Superoxide dismutase activity and the effect of N-methyl-D-aspartate antagonists on lipid peroxidation in the early phase of cold injury.

Free radicals, lipid peroxidation and excitatory amino acids have been implicated in the secondary mechanisms of traumatic brain injury. We used the cold injury model in rats to assess the endogenous activity of the protective enzyme superoxide dismutase (SOD) and the lipid peroxidation level in the contused tissue at an early phase of injury. Furthermore, we treated the rats with two different N-methyl-D-aspartate receptor antagonists, namely MK-801 and CPP, and evaluated their effect on lipid peroxidation in the contused tissue. Rats were divided into four groups: sham, control, treatment 1 and treatment 2 groups (n= 16 for each group). Thirty and 60 min after craniectomy or injury, tissue samples were removed. SOD activity didn't change in this period. However, lipid peroxidation in terms of malondialdehyde (MDA) amount showed a significant increase at 60 min. Fifteen minutes after injury, MK-801 (1 mg/kg), CPP (10 mg/kg) or saline (1 ml) were applied intraperitoneally in treatment 1, treatment 2 and the control groups. Treatment with MK-801 attenuated MDA levels, whereas treatment with CPP did not. The protective effect of MK-801 achieved statistical significance. These results demonstrate that SOD activity does not change in the early period of cold injury. Moreover, these results show that lipid peroxidation increases after 60 min of cold injury, and treatment with MK-801 15 min after injury can prevent this elevation.

Expression of nitric oxide synthase in the cerebral microvasculature after traumatic brain injury in the rat.

Reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) histochemistry and nitric oxide synthase (NOS) immunocytochemistry were performed on sections of brain after moderate traumatic brain injury. There was a pronounced increase in NADPHd reactivity and an induction of the endothelial NOS (eNOS) isoform in microvessels surrounding the cortical contusion by 24 h post-injury. This altered microvascular state may contribute to barrier breakdown and hyperemia which characterize traumatic brain injury.

Induction of Cu,Zn-superoxide dismutase after cortical contusion injury during hypothermia.

To determine the effect of hypothermia on superoxide injury after cerebral contusion, the induction of Cu,Zn-superoxide dismutase was examined 6 h after contusion in rats using Northern blotting. Cu,Zn-superoxide dismutase gene expression increased at the periphery of the contusion, which may indicate the severity of the superoxide stimulus. This increase was preserved after contusion under hypothermia, which may show that superoxide injury is still severe although brain edema is decreased.

[The superoxide dismutase activity of the brain and liver tissues in a rabbit with an experimental brain concussion]. / Aktivnost' superoksiddismutazy v tkaniakh mozga i pecheni krolika pri sotriasenii mozga v éksperimente.

A study is presented of the activity of superoxidedismutase and catalase in the brain and liver in the dynamics of mild head injury in experiments on rabbits. It was established that the activity of catalase in the brain show a three-times reduction within 15 minutes after the injury. The catalase activity in the liver remains practically unchanged. The activity of superoxidedismutase in the liver in the posttraumatic period does not differ from control values. In the brain superoxidedismutase activity reduces by 38% only on the 7 and 14 days after injury.

Serum creatine kinase isoenzyme BB is a poor index to the size of various brain lesions.

We divided patients with brain lesions into three groups: (a) patients with primary or metastatic brain cancer, (b) brain infarctions, and (c) brain contusion(s). We analyzed each patient's sera for creatine kinase isoenzyme BB (CK-BB), using a monoclonal antibody kit (Impres-BB; International Immunoassay Laboratories). Computerized axial tomography (CAT) scans were performed on each patient. The size of the various lesions was measured from the CAT scan and recorded in milliliters. Total CK, CK-BB, and their ratios were compared with the volume of damaged brain tissue. We found no correlation between any of the variables and the various brain lesions. We attribute this lack of correlation to an intact blood-brain barrier, the rapid elimination or inactivation of CK-BB, or some combination of these factors.

[Evaluation of the activity of various lysosomal enzymes in the cerebrospinal fluid and blood in cases of brain concussion]. / Ocena aktywnosci niektórych enzymów lizosomalnych w plynie mózgowo-rdzeniowym i krwi w przypadkach stluczenia mózgu.

The results of determinations of lysosomal enzymes: mannosidase, b-galactosidase and b-glucosidase, in the cerebrospinal fluid and blood are presented in 72 patients with severe brain trauma. The determinations were carried out at different times after the trauma. The study demonstrated a significantly lower level and in a smaller degree expressed variability of enzymes in the cerebrospinal fluid in relation to blood. It seems that the level of b-glucosidase in the cerebrospinal fluid depends on the level of this enzyme in the blood, while no such evident correlation was found for the remaining enzymes.

An assessment of acid-base balance and activity of lysosomal enzymes: mannosidase, b-galactosidase and b-glucosidase in CSF and arterial blood in early and later phases of the clinical course of cerebral contusion.

Results of the examination of the acid-base balance and the concentration of enzymes - mannosidase, b-galactosidase and b-glucosidase in 61 patients with cerebral contusion are presented. Examination was carried out on an average 11, 15 and 39 days after injury in groups of patients characterised by various clinical states. Examination showed that, in contradistinction to the acid-base balance, enzymal changes in CSF are less evident than in blood. On the base of the results presented it would appear that the level of b-glucosidase in CSF changes in accordance with the level in the blood whereas the other enzymes show a certain autonomy of the CSF in this field.

Creatine kinase BB in blood as index of prognosis and effect of treatment after severe head injury.

High concentrations of creatine kinase BB (CK-BB) were found in all blood samples drawn within 6 hours of accident from 45 patients with brain contusion. The highest concentrations of more than 100 microg/l were measured in blood samples taken shortly after the accident from patients with a Glasgow Coma Score (GCS) of 6 or less. The CK-BB concentrations decreased rapidly to normal within 36 hours of accident in the patients given intensive care guided by intracranial pressure (ICP) monitoring. In patients with less severe injuries according to GCS the initial CK-BB concentrations were generally lower and normalized less rapidly. The outcome after 6 months was moderate or good in all 9 patients who had this rapid normalization of blood CK-BB. On the other hand, of 20 patients who had a more slow CK-BB decrease, only 9 had an acceptable outcome. Delayed ICP increase to more than 40 mm Hg and even delayed brain tamponade did not result in CK-BB levels higher than 5 microg/l. Brain tamponade in the acute stage resulted in rapid CK-BB decrease in the blood. In paired simultaneously drawn samples of lumbar cerebrospinal fluid (CSF) and blood, CK-BB levels were generally higher in the CSF.

The prognosis of patients with concussion and increased creatine kinase BB in the cerebrospinal fluid.

In a series of 93 emergency patients, 58 were classified as concussions on a clinical basis. Thirteen of the patients with concussion had increased levels of creatine kinase isoenzyme BB (CK-BB) in their cerebrospinal fluid (CSF). We performed a prospective, follow-up investigation comparing 10 patients with a CK-BB increase and 10 patients without a CK-BB increase after concussion. Within 24 hours, at 6 months and 3 years after concussion, each patient was subject to a special interview to obtain pre-concussional baseline data and post-concussional follow-up data concerning their complaints and capacity for daily activities. We found a definite change towards increasing disability in 8 of the 10 patients with a raised CK-BB, and in only 1 of the 10 patients with normal levels of CK-BB. A careful neuropsychological examination confirmed inferior performance in tests especially sensitive to brain injury in patients with a CK-BB increase. Our results suggest that increased levels of CK-BB after concussion signify a more severe injury which is not found in the clinical examination during the first days after the accident, and that these patients are a high-risk group for the development of post-concussional problems and symptoms.