[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.

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.

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.

[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.

[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.

Hyperamylasemia: a result of intracranial bleeding.

The unexpected finding of hyperamylasemia in a patient with isolated head injury prompted a study of amylase levels in patients with various degrees of cranial trauma. None of seven patients with isolated maxillofacial trauma had hyperamylasemia. This group was studied to discount injury to salivary glands as a source of elevated amylase levels. Only one of ten patients with simple cranial injury without computerized tomographic (CT) scan evidence of intracranial bleeding had hyperamylasemia. Six of ten patients with CT scans positive for intracranial bleeding had hyperamylasemia. Isoamylase analysis showed that the source of the hyperamylasemia was varied. These results suggest a central neural control of serum amylase levels. The reliability of the serum amylase level as an indication of pancreatic trauma in a patient with concomitant head injury is questioned.

Plasma concentrations of granulocytic elastase-alpha 1-proteinase inhibitor complex in patients with severe head injury, multiple trauma or cerebral bleeding.

In patients with severe head injury or multiple trauma a multitude of inflammatory mediators are released. As in cerebral bleeding, elevations of body temperature can be observed even in the absence of bacterial infections. In order to evaluate the diagnostic significance of plasma elastase levels for the exclusion or early detection of infections in the course of the above diseases, quantitative estimations of granulocytic elastase in complex with alpha 1-proteinase inhibitor were carried out in 11 patients using an enzyme-linked immunoassay over a period from 3 to 47 days. Additionally, leukocyte counts were made and body temperatures measured. In all but two cases, elastase levels were elevated at the beginning of the disease and usually decreased over the period of study. In contrast to leukocyte counts and body temperature, the values correlated with the severity of the clinical symptoms. It is concluded that plasma elastase determinations are useful in the differential diagnosis of infection from other causes of elevated body temperatures.

Modifications of plasma enzyme activities after severe head injury; evaluation of prognosis using multivariate methods.

The aim of this study was to analyse the plasma variations of four enzymatic activities (lactate dehydrogenase, aldolase, creatine kinase, aspartate aminotransferase) in 134 patients suffering from severe head injury. Enzymatic activities were assayed daily for 3 days after the trauma. Means of the four enzymatic activities were significantly different according to their evolution (death or survival), except for creatine kinase, 48 h after the trauma. Multivariate analysis indicated that lactate dehydrogenase and aldolase levels were useful in order to discriminate between potential survivors and non-survivors. The value of multivariate analysis in head traumatology is discussed.

Severe head trauma and the changes of concentration of neuron-specific enolase in plasma and in cerebrospinal fluid.

In nine patients with severe head trauma, the concentration of neuron-specific enolase in cerebrospinal fluid and in plasma was determined and compared with the activity of creatine kinase and alpha-hydroxybutyrate dehydrogenase, and with the concentration of lactate. In patients who died of the head trauma, a concentration of neuron-specific enolase of 6.8-64 micrograms/l in the plasma (reference range: 3.0-6.0 micrograms/l) and of 2.2-9.0 micrograms/l in the cerebrospinal fluid (reference range: 0.5-2.0 micrograms/l) was detected. Investigations of three patients showed that the changes of the concentration of neuron-specific enolase in plasma and in cerebrospinal fluid were independent of each other. Furthermore, the initial concentration of neuron-specific enolase in the plasma after the accident and the dynamics of its changes during the disease show a close relationship to the outcome.

CSF brain creatine kinase levels and lactic acidosis in severe head injury.

The posttraumatic creatine kinase-BB isoenzyme (CKBB) activity and lactate concentration in ventricular cerebrospinal fluid (CSF) have been studied in 29 patients with severe head injuries. The CKBB activity reaches its maximum a few hours after trauma, and has a monoexponential drop with a half-time of approximately 10 hours. Ventricular CSF lactate concentration continues to rise in patients with a poor outcome, and decreases only slowly and inconsistently in most of the other patients. Thus, increase of lactate in the ventricular CSF is not, like CKBB, a direct one-stage consequence of the trauma but is due to continuous production from a derangement of metabolism caused by the trauma. Since even higher ventricular CSF lactate levels can be survived when not caused by head injury, and since no significant pH changes were related to the ventricular CSF lactic acidosis in these artificially ventilated patients, it is concluded that ventricular CSF lactic acidosis is indicative of a severe, although not necessarily intractable, disturbance of brain function associated with intracellular lactate production and acidosis.

Biochemical and biophysical changes in guinea pigs after acute head injury.

Animal experiments were set up mainly to derive additional diagnostic data from the study of biochemical changes after acute head injury. In standardized experiments guinea pigs were subjected in groups of 20 to three identical head injuries, each of either 1.0 J or 1.5 J intensity. The trauma was likely to result in a concussion or contusion syndrome similar to that found in man; 40 animals served as controls. During the 60 min after injury observation and measurement of body functions did not reveal signs of a shock-like condition or hypoxaemia in the traumatized animals compared with control animals. Superficial anaesthesia probably did not influence the findings. Temperature and respiration were altered significantly in all the animals receiving head injuries. Blood gas analysis showed a decrease of standard bicarbonate only after the 1.5 J injury but even though hypoxaemia was not present 2,3-diphosphoglycerate values and P50 increased, compared with the control animals. The fall of plasma lipid concentrations reported probably had to be seen as a sympathomimetic effect of the minor (1.0 J) trauma. Of special significance was the increased activity of malate dehydrogenase and aldolase, found only in the blood of severely traumatized animals, as this could serve as an early diagnostic aid for evaluating head injuries.

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.

Amylase isoenzymes in the evaluation of trauma patients.

In traumatized patients, elevation of the levels of serum amylase is often noted and may lead to a diagnosis of pancreatitis or pancreatic injury. In the presence of multiple injuries, it is often difficult to evaluate clinically for pancreatitis or pancreatic injury. Since the serum amylase is derived from both the pancreatic and the salivary glands, it is useful to determine the origin of the elevated levels of serum amylase in these patients. A total of 31 patients including 21 trauma patients were studied, and the total serum amylase and also the pancreatic (P) and salivary (S) fractions were determined by isoelectric focusing. Compared with the normal control group, most trauma victims had elevated total amylase levels (normal, 30-128 U). In six patients with head and facial trauma, the P-fraction was 7.6 per cent, and the S-fraction was 92.4 per cent (normal, P 35-50%; S 50-65%), while in six patients with penetrating abdominal trauma, the P-fraction was 81 per cent, and the S-fraction was 19 per cent. These differences were statistically significant. The data demonstrate the value of measuring fractions of amylase in addition to total amylase levels. In patients with head and facial trauma alone, elevated levels of serum amylase are due to an increase in the salivary fraction. Elevation of total serum amylase in traumatized patients does not necessarily indicate pancreatic injury. Measurements of amylase fractions were thus useful in evaluation of trauma patients.

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.

Brain protease activity after experimental head injury.

In an experimental series on twelve cats, activity changes of brain cell proteolytic activity were measured two hours after a blunt head injury without hematoma or contusions. Protease activity was estimated in two different brain tissue homogenate supernatants containing total soluble and only cytoplasmic activity without proteases in cell organelles, respectively. Total activity was doubled two hours after injury in the acid and the neural pH-range, in comparison to control values. Free soluble activity was doubled in the acid and increased to the threefold value in the neutral range. From these data, it seems that two different changes appear in lysosomes, the enzyme-reservoir of the cell: (1) Enzyme-synthesis is increased after trauma, measured as augmentation of total soluble protease activity in our experiments; (2) Breakage or increased permeability of lysosomes lead to augmentation of especially neutral proteases in the cytoplasm followed by the well known autolytic areas of generalized traumatic brain edema.

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.