Distinct roles for metalloproteinases during traumatic brain injury.

BACKGROUND: Significant protease activations have been reported after traumatic brain injury (TBI). These proteases are responsible for cleavage of transmembrane proteins in neurons, glial, and endothelial cells and this results in the release of their extracellular domains (ectodomains). METHODS: Two TBI models were employed here, representing both closed head injury (CHI) and open head injury (OHI). In situ zymography, immunohistochemistry, bright field and confocal microscopy, quantification of immunopositive cells and statistical analysis were applied. RESULTS: We found, using in situ zymography, that gelatinase activity of matrix metalloproteinases (MMP)-2 and MMP-9 was upregulated in cortex of both injury models. Using immunohistochemistry for several MPPs (Matrix metalloproteinases) and ADAMs (disintegrin and metalloproteinases), including MMP-2, -9, ADAM-10, -17, distinct patterns of induction were observed in the two TBI models. In closed head injury, an early increase in protein expression of MMP-2, -9 and ADAM-17 was found as early as 10 min post injury in cortex and peaked at 1 h for all 4 proteases examined. In contrast, after OHI the maximal expression was observed locally neighboring the impact site, at a later time-point, as long as 24 h after the injury for MMP-2 and MMP-9. Confocal microscopy revealed colocalization of the 4 proteases with the neuronal marker NeuN in CHI, but only MMP2 colocalized with NeuN in OHI. CONCLUSIONS: The findings may lead to a trauma-induced therapeutic strategy triggered soon after a primary insult to improve survival and to reduce brain damage following TBI.

[Clinical and prognostic significance of genetic markers in craniocerebral injury (Part III)].

It is now becoming increasingly clear that the course and outcome of craniocerebral injury (CCI) are determined not only by its biomechanism, severity, patient's age, presence of premorbid factors, etc., but also by individual features of the genome of each patient, which puts traumatic brain injury among multifactorial diseases. The genome determines the presence or absence of«genetic predilection to the development of various complications and sequelae of CCI, which generally determines the progression of traumatic brain injury disease. The first part of the review by Potapov et al. (201 0) [2] was devoted to the role of apolipoprotein E (apoE) gene polymorphism in CCI, the second one [3]- to the role of inflammation and immune response genes in the course and outcome of CCI. The present (third) part will provide a review of modern data on the effect of genes underlying intracellular processes of oxidative stress, apoptosis, regeneration, and synthesis of neurotransmitters and their receptors.

Study of the neuroprotective effect of ginseng on superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels in experimental diffuse head trauma.

BACKGROUND: The purpose of our study was to investigate the effect of ginseng on antioxidant enzyme levels in brain damage following experimental diffuse head trauma in rats. The neuroprotective effect of ginseng was also studied. METHODS: In this study, rats were divided into four groups, and the rats in group 1 received no intervention. In group 2, the rats were administered 50 mg/kg ginseng, injected intraperitoneally at 1, 24 and 48 h, and the effect of ginseng on normal tissues was studied. No drugs were administered to the rats in group 3 who had previously experienced diffuse head trauma using Feeney's falling weight method. In group 4, rats underwent Feeney's falling weight method, leading to diffuse head trauma, and they were given 50 mg/kg ginseng intraperitoneally 1, 24 and 48 h after head trauma. Rats were killed 72 h after head trauma and their brain tissues extracted for histopathological and biochemical studies. RESULTS: Histopathological study of brain cross sections in the trauma group demonstrated neurons in the trauma region and surrounding area, which generally had a dark-colored eosinophilic cytoplasm and a pyknotic nucleus, while the nuclei of neurons were located peripherally. However, brain cross sections in group 4 from rats given ginseng after head trauma showed fewer neurons with eosinophilic cytoplasm, pyknotic and peripheral nuclei in the trauma region and surrounding area. No statistically significant difference in the tissue SOD level was observed; however, the GSH Px level in group 4 was significantly reduced compared to that in group 3. CONCLUSIONS: After affecting the GSH Px level and reducing histopathological scores, ginseng was found to display antioxidant and neuroprotective activity.

The release of S-100B and NSE in severe traumatic head injury is associated with APOE ε4.

OBJECT: In this article we tested the hypothesis that the level of two biochemical markers of brain injury may be associated with the apolipoprotein E (APOE) ε4 allele. METHODS: In this prospective consecutive study patients with sTBI were included (n = 48). Inclusion criteria were Glasgow Coma Scale (GCS) score ≤ 8 at the time of intubation and sedation, patient age between 15 and 70 years, an initial cerebral perfusion pressure > 10 mmHg, and arrival to our level-one trauma university hospital within 24 h after trauma. Blood samples for neuron-specific enolase (NSE) and S-100B were collected as soon as possibly after arrival, and then twice daily (12-h intervals) for 5 consecutive days. Venous blood was used for APOE genotype determination. Clinical outcome at 3 months after injury was assessed with the Extended Glasgow Outcome Scale (GOSE). RESULTS: Significantly higher levels of the maximal S-100B (S-100B(max)) and area under the curve (S-100B(AUC)) were found in subjects with the APOE ε4 allele compared to those with non-ε4. A similar tendency was observed for NSE(max) and NSE(AUC), though not statistically significant. CONCLUSION: Our data indicate that there might be a gene-induced susceptibility to severe traumatic brain injury and that patients with the APOE ε4 allele may be more predisposed to brain cellular damage measured as S-100B and NSE. Thus, it seems to be of importance to consider the APOE genotype in interpreting the levels of the biomarkers.

The protective effects of propofol and citicoline combination in experimental head injury in rats.

AIM: Lipid peroxidation (LP) is an important factor in tissue damage following head injury. Reactive oxygen radicals which damage cellular components play an important role in ischemic or hypoxic tissue. They initiate the lipid peroxidation process after head trauma. However, antioxidant agents may protect brain tissue against oxidative damage MATERIAL AND METHODS: 39 male Swiss Albino rats (200-250 g) were used in this experimental study. These animals were divided into 3 groups: 1) control group, 2) propofol group (100 mg/kg) and, 3) citicoline (250 mg/kg) and propofol (100 mg/kg) combination group. Oxidant effect in brain tissue content was assessed by measuring the Malonyldialdehyde (MDA), Superoxide Dismutase (SOD) and Gluthatione Peroxidase (GPx) activities. RESULTS: There was no statistically meaningful difference among the groups regarding GPx levels. MDA levels were significantly lower in the citicoline and combination group than those of the control group. As for the levels of SOD, there was an increase both in the propofol and combination groups. CONCLUSION: Atherapeutic benefit of the propofol and citicolin combination in head trauma has not been previously demonstrated. We examined the possible potential protective effect of propofol and citicolin against oxidative damage in experimental head trauma in the present study.

Quantitative analysis and localization of mRNA transcripts of type I collagen, osteocalcin, MMP 2, MMP 8, and MMP 13 during bone healing in a rat calvarial experimental defect model.

The study examined the expression of matrix metalloproteinases (MMPs), type I collagen and osteocalcin during bone healing in a rat calvarial experimental defect model. Twelve-week-old male Wistar rats were used. A full-thickness standardized trephine defect was made in the parietal bone, with the rat under anesthesia. RNA was extracted from tissue that filled the original bone defect on days 1 and 3 and in weeks 1, 2, 3, 5, 8, 10, 12, 18, and 24 and processed for quantitative analysis of expression of type I collagen, osteocalcin and matrix metalloproteinases (MMPs) 2, 8, and 13 by using real-time polymerase chain reaction. Alternatively, the rats were fixed by perfusion through the aorta and resected calvaria were processed for in situ hybridization for these molecules. The expression of type I collagen, osteocalcin and MMPs 2 and 13 increased toward week 2 and decreased thereafter, whereas the expression of MMP 8 was the highest on day 1. The mRNA transcripts of type I collagen and osteocalcin were localized in osteoblasts and osteocytes, some of which expressed MMPs 2, 8, and 13. Osteoblasts and osteocytes may play a role in the remodeling of extracellular matrices with MMPs during healing of a defect in bone.

[Changes of brain p38 MAPK in rabbits with craniocerebral gunshot injury in hot and humid environment].

OBJECTIVE: To study the activation of p38 mitogen-activated protein kinase (MAPK) in the brain of rabbits after craniocerebral gunshot injury in a hot and humid environment (HHE) and explore its possible mechanism. METHODS: Craniocerebral gunshot injury model was established in 30 New Zealand white rabbits, which were subsequently exposed to environment of normal temperature (at 22.0% +/- 0.5 degrees C; with relative humidity of 50%) or HHE at 39.0 +/- 0.5 degrees C; with relative humidity of 80%-85% for 10 min, 30 min, 1 h, 1.5 h, and 2 h groups, respectively, with 5 rabbits in each group. p38 MAPK activity in the brain tissues of the rabbits following the injury and environmental exposure were detected by Western blotting and analyzed semi-quantitatively by Bio-Profil gel image analysis system. RESULTS: p38 MAPK activity in the cortex and hypothalamus was significantly elevated following gunshot injury and HHE exposure, reaching the peak level at 1 h of HHE exposure and then decreased. p38 MAPK activity was significantly higher in the hypothalamus than in the cortex. CONCLUSION: p38 MAPK activity increases in the early stage following craniocerebral gunshot injury and HHE exposure in rabbits, the mechanism of which might involve the secondary brain insult.

[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.

[Effects of propofol and isoflurane on serum neuron-specific enolase level in surgical patients with acute craniocerebral trauma: a comparative study].

OBJECTIVE: To investigate changes in serum neuron-specific enolase (NSE) concentration in patients before, during and after surgery for acute craniocerebral trauma, and examine the effects of propofol and isoflurane on these changes. METHODS: Ten patients scheduled for urinary operation without cerebral injury were enrolled in the control group. Thirty patients with acute cerebral trauma were randomly allocated to propofol group (n=15) and isoflurane group (n=15). Serum concentrations of NSE before surgery, 2 h after the surgery began, and after completion of surgery were measured in all the patients by enzyme-linked immunoadsorbent assay. Glasgow scores of patients with cerebral trauma were also estimated and recorded. RESULTS: Serum concentration of NSE in patients with cerebral trauma were significantly higher than those in the control group before the surgery (P<0.01). The Glasgow score was inversely correlated with serum NSE concentration (r=-0.494, P<0.01). Serum NSE level after completion of surgery was significantly lower in propofol group than in isoflurane group (P<0.05). NSE levels at 2 h after the initiation of the surgery and after the completion of surgery were higher than those before the surgery. CONCLUSIONS: Serum NSE concentration increases in patients with acute cerebral injury in parallel to the severity of brain damage. Application of propofol by intravenous pumping can reduce the increase in serum NSE, alleviate cerebral injury, and protect the brain tissues of patients undergoing surgery for acute craniocerebral trauma.

Apoptotic change and NOS activity in the experimental animal diffuse axonal injury model.

Although nitric oxide (NO) plays an important role in the pathophysiological process of cerebral ischemia or severe traumatic brain injury, its contribution to the pathogenesis of moderate diffuse axonal injury (mDAI) remains to be clarified. The alterations in nitric oxide synthase (NOS) activity and the histopathological response after mDAI was investigated. Forty anesthetized Sprague-Dawley adult rats were injured with a Marmarou's weight-drop device through a Plexiglas guide tube. These rats were divided into 8 groups (control, 1 hr, 2 hr, 3 hr, 6 hr, 12 hr, 24 hr, 48 hr after trauma). The temporal pattern of apoptosis in the adult rat brain after mDAI was characterized using TUNEL histochemistry. In addition, the cDNA for NOS activity was amplified using RT-PCR. The PCR products were electrophoresed on a 2% agarose gel. eNOS activity was not detected, but nNOS activity was expressed after 3 hr and continuously 48 hr after impact, which was approximately double that of the control group at 12 and 24 hr. Subsequently, there was a decrease in activity after 48 hr. The iNOS activity increased dramatically after 12 hr and was constant for a further 12 hr followed by a dramatic decrease below the level of the control group. Significant apoptotic changes occurred 12 and 24 hr. after insult. nNOS and iNOS activity were affected after moderate diffuse axonal injury in a time-dependent manner and there was a close relation between the apoptotic changes and NOS activity. Although the nNOS activity was expressed early, its activity was not stronger than iNOS, which was expressed later.

A new stroke marker as detected by serum phosphoglycerate mutase B-type isozyme.

The diagnostic significance of phosphoglycerate mutase (PGM) B-type isozyme activity capable of being inducible under hypoxia at the gene level as a serum marker for cerebral stroke was investigated. The normal level (mean +/- 2 SD) in human serum was determined to be 38 +/- 18 units/L. Within 2 h after the onset of cerebral stroke (n = 65), B-type isozyme activity was elevated to 68 +/- 36 units/L, and retained to be higher level until 1-3 days. Serum B-type isozyme activities of 52 survival cases and 13 dead cases, being judged at the period of 1-2 months after the onset, were retrospectively compared; B-type isozyme activity that had been measured within 24 h after the onset was significantly higher (81 +/- 42 units/L) for the dead cases than for survival cases (57 +/- 27 units/L) with P < 0.05. These results suggest that serum PGM B-type isozyme has the potential as a novel marker for diagnosis of cerebral stroke and its severity.

Serum amylase and lipase elevation is associated with intracranial events.

Serum amylase and lipase elevation has been observed in trauma patients and patients with traumatic intracranial bleeding. However, the causes of this elevation have not been clearly elucidated. A further question remains as to whether other intracranial events are associated with such enzyme elevation as well. We retrospectively reviewed 75 patients consecutively admitted to Cook County Hospital Neurosurgical Intensive Care Unit over a 3-month period for trauma, infection, tumor, or other space-occupying lesions with an unstable condition or neurological deficit. Eleven patients (15%) had elevated amylase and lipase levels. The patients were divided into two groups: Group I (n = 64) had normal and Group II (n = 11) had raised amylase and lipase levels [amylase 402 +/- 444 U/L with normal < or = 125 U/L and lipase 474 +/- 313 U/L with normal < or = 55 U/L]. All Group II patients suffered an intracranial event. Twenty-four Group I (38%) and 10 Group II (91%) patients required craniotomy (P < 0.01). No patients had clinical or radiographic evidence of pancreatitis. In summary, intracranial events are associated with serum amylase and lipase elevation probably through centrally activated pathways. Because of the lack of diagnostic value, routine pancreatic enzyme monitoring should not be performed in this patient population.

Apoptotic Change and NOS Activity in the Experimental Animal Dif fuse Axonal Injury Model

Although nitric oxide (NO) plays an important role in the pathophysiological process of cerebral ischemia or severe traumatic brain injury, its contribution to the pathogenesis of moderate diffuse axonal injury (mDAI) remains to be clarified. The alterations in nitric oxide synthase (NOS) activity and the histopathological response after mDAI was investigated. Forty anesthetized Sprague-Dawley adult rats were injured with a Marmarou's weight-drop device through a Plexiglas guide tube. These rats were divided into 8 groups (control, 1 hr, 2 hr, 3 hr, 6 hr, 12 hr, 24 hr, 48 hr after trauma). The temporal pattern of apoptosis in the adult rat brain after mDAI was characterized using TUNEL histochemistry. In addition, the cDNA for NOS activity was amplified using RT-PCR. The PCR products were electrophoresed on a 2% agarose gel. eNOS activity was not detected, but nNOS activity was expressed after 3 hr and continuously 48 hr after impact, which was approximately double that of the control group at 12 and 24 hr. Subsequently, there was a decrease in activity after 48 hr. The iNOS activity increased dramatically after 12 hr and was constant for a further 12 hr followed by a dramatic decrease below the level of the control group. Significant apoptotic changes occurred 12 and 24 hr. after insult. nNOS and iNOS activity were affected after moderate diffuse axonal injury in a time-dependent manner and there was a close relation between the apoptotic changes and NOS activity. Although the nNOS activity was expressed early, its activity was not stronger th an iNOS, which was expressed later.

Neutrophil elastase may play a key role in developing symptomatic disseminated intravascular coagulation and multiple organ failure in patients with head injury.

OBJECTIVE: To study the mechanism associated with the development of symptomatic disseminated intravascular coagulation (DIC) after head injury. METHODS: Plasma parameters were analyzed in patients with symptomatic (group A, n = 10) and asymptomatic DIC (group B, n = 15) induced by head injury, and in patients in whom DIC was caused by sepsis (group C, n = 10). RESULTS: Levels of fibrinogen, alpha2PI-plasmin complex and platelets in group A (58.1 mg/dL, 22.4 microg/mL, 16.0 x 10(4)/ mm3) and group B (98.3, 22.1, 16.6 x 10(4)) were comparable, but differed significantly from those in group C (297.4, 2.4, 6.3 x 10(4)). Significant differences were observed between groups A and B in both neutrophil-elastase (1,528 vs. 293 microg/ml) and D-dimer (42.1 vs. 17.6 microg/mL). CONCLUSION: Neutrophil elastase may be implicated in the development of symptomatic DIC after head injury, whose characteristics include "enhanced fibrinolysis with minimal platelet loss."

Prognostic value of serum neuron-specific enolase levels after head injury.

Neuron-specific enolase (NSE) is a dimeric cytoplasmic enzyme detected in high levels in neurons and acts in the glycolytic pathway. It is known that there is a quantitative relationship between the concentration of serum NSE and the degree of cell damage in the central nervous system. We examined serum levels of NSE by enzyme immunoassay in 89 patients with head injury and aimed to evaluate its relationship with neurological status and prognosis of the patients.

Decreasing the apoptotic threshold of tumor cells through protein kinase C inhibition and sphingomyelinase activation increases tumor killing by ionizing radiation.

Approximately 30% of cancer deaths result from the failure to control local and regional tumors. The goal of radiotherapy is to maximize local and regional tumor cell killing while minimizing normal tissue destruction. Attempts to enhance radiation-mediated tumor cell killing using halogenated pyrimidines, antimetabolites, and other DNA-damaging agents or sensitizers of hypoxic tumor cells have met with only modest clinical success. In an unique strategy to modify tumor radiosensitivity, we used an inhibitor of the protein kinase C group A and B isoforms, chelerythrine chloride (chelerythrine), to enhance the killing effects of ionizing radiation (IR). Protein kinase C activity plays a central role in cellular proliferation, differentiation, and apoptosis. Chelerythrine increases sphingomyelinase activity and enhances IR-mediated cell killing through induction of apoptotic tumor cell death in a radioresistant tumor model both in vitro and in vivo. Although previous reports have suggested that IR-mediated apoptosis correlates with tumor volume reduction, we demonstrate for the first time that lowering the apoptotic threshold increases tumor cell killing in vivo.

Serum pancreatic enzymes in patients with coma due to head injury or acute stroke.

Serum amylase and lipase were measured in 32 patients with cerebral ischemia, 19 with spontaneous cerebral hemorrhage, 15 with head injury and intracranial bleeding, and 22 with head injury without intracranial bleeding; 20 healthy subjects were also studied as controls. Serum pancreatic isoamylase concentrations were assayed in hyperamylasemic sera. The overall incidence of hyperamylasemia was 14% (12 of 88 patients: 4 with cerebral ischemia, 4 with spontaneous cerebral hemorrhage, 1 with head injury and intracranial bleeding, and 3 with head injury without intracranial bleeding). In 4 of the 12 patients the hyperamylasemia was of pancreatic origin: 1 patient with cerebral ischemia, 1 patient with spontaneous cerebral hemorrhage, 1 patient with head injury and intracranial bleeding, and 1 patient with head injury without intracranial bleeding. The incidence of hyperlipasemia was 7% (6 of the 88 patients: 1 patient with cerebral ischemia, 2 with spontaneous cerebral hemorrhage, and 3 with head injury without intracranial bleeding). We conclude that hyperamylasemia is more frequent than hyperlipasemia in patients with an altered state of consciousness due to head injury or stroke and is usually of non-pancreatic origin. This knowledge may save these patients from invasive and costly examinations.

Significance of elevated pancreatic enzymes in intracranial bleeding.

Hyperamylasemia of pancreatic origin has been noted in patients with severe head injury without abdominal trauma or evidence of pancreatitis. Thirty-eight patients with intracranial bleeding of various types were evaluated for elevated pancreatic amylase and lipase enzymes without associated pancreatitis. Twenty-five patients had elevated serum lipase; 17 of 25 also had elevated amylase without pancreatitis. Most lipase elevations occurred earlier than those of amylase. Six clinical variables--mannitol, ceftriaxone, nimodipine, steroids, Glasgow Coma Score, and total parenteral and enteral hyperalimentation--were evaluated to determine relationship to the enzyme elevations. A significant relationship exists between patients not treated with steroids and elevated lipase and amylase enzyme activities. Multivariate analysis revealed a significant interaction between lipase elevation and decreasing Glasgow Coma Score, indicative of increasing severity of intracranial bleeding. Proposed causes of enzyme elevations in intracranial bleeding include vagal stimulation, altered modulation of the central control of pancreatic enzyme release, and release of cholecystokinin from the brain. Physician awareness of the association of intracranial bleeding with the elevation of amylase and lipase without pancreatitis can save the patient needless cost and manipulation.

The prognostic value of the brain sodium-potassium ATPase enzyme concentration in head injury.

Sodium-potassium adenosine triphosphatase (ATPase) enzyme was determined in the brain tissue of 11 patients with head injury and 6 control patients. Patients with head injury included in this study were selected from two categories: (a) patients in deep coma due to severe head injury [Glasgow Coma Scale (GCS) less than 8; 6 cases]; (b) patients with depressed skull fractures with dural tears who were conscious and able to give an adequate verbal response (GCS greater than 10; 5 cases). The level of the enzyme was significantly reduced in comatose patients with severe head injury as compared to the controls (P less than 0.001) or to conscious patients with depressed fractures (P less than 0.001). In the group of conscious patients with depressed fractures, the enzyme level was no different from that of the controls (P = 0.4215). All comatose patients with severely reduced enzyme levels subsequently died, whereas those with depressed fractures with normal enzyme levels survived. The relationship between a low enzyme level and brain edema in severe head injury is discussed.

[Clinical studies on hypercreatine phosphokinasemia in emergency diseases].

Unexplained serum creatine phosphokinase (CPK) elevation is not a rare clinical problem, especially in emergency diseases. We studied hypercreatine phosphokinasemia (hyper-CPK-emia) in 161 cases of emergency diseases. Correlations between CPK and various laboratory data, various conditions were investigated. The hyper-CPK-emia was found to obtain no correlations with GPT, GOT, LDH, creatinine and body temperature. For this reason, we could not discover the unknown factors of which contributed to elevate the serum CPK. The sources of the serum CPK in these diseases were concluded to be the skeletal muscle. This is based on the facts that CPK MM (muscle type) is specifically increased among the CPK isozymes. The elevation of serum CPK activity in emergency diseases was considered to result from muscle hypoxia due to severe stress and general circulatory failure.