Andreas Kampfl

Prediction of recovery from post-traumatic vegetative state with cerebral magnetic-resonance imaging.

BACKGROUND The early post-traumatic vegetative state (VS) is compatible with recovery. Various clinical and laboratory tests have failed to predict recovery so we assessed the value of cerebral magnetic-resonance imaging (MRI) in prediction of recovery. METHODS 80 adult patients in post-traumatic VS had cerebral MRI between 6 weeks and 8 weeks after injury. MRIs were reviewed by three neuroradiologists for the number, sizes, and location of brain lesions. Three neurologists assessed the patients at the time of MRI and at 2 months, 3 months, 6 months, 9 months, and 12 months after injury using the Glasgow Outcome Scale. FINDINGS At 12 months, 38 patients had recovered while 42 patients remained in the VS. The demographic characteristics and causes and severity of injury were similar in patients in persistent VS (PVS) and those who recovered (NPVS). An average of 6.1 different brain areas were injured in patients in PVS compared with 4.6 areas in patients who had NPVS. Patients in PVS revealed a significantly higher frequency of corpus callosum, corona radiata, and dorsolateral brainstem injuries than did patients who recovered. Logistic regression analysis showed that corpus callosum and dorsolateral brainstem injuries were predictive of non-recovery. The adjusted odds ratios for non-recovery of patients with a corpus callosum lesion and dorsolateral brainstem injury were 213.8 (95% CI 14.2-3213.3), and 6.9 (11-42.9), respectively. In contrast, clinical characteristics, such as initial score on the Glasgow Coma Scale, age, and pupillary abnormalities failed to predict recovery. INTERPRETATION Cerebral MRI findings in the subacute stage after head injury can predict the outcome of the post-traumatic VS. Corpus callosum and dorsolateral brainstem lesions are highly significant in predicting non-recovery.

A calpain inhibitor attenuates cortical cytoskeletal protein loss after experimental traumatic brain injury in the rat

The capacity of a calpain inhibitor to reduce losses of neurofilament 200-, neurofilament 68- and calpain 1-mediated spectrin breakdown products was examined following traumatic brain injury in the rat. Twenty-four hours after unilateral cortical impact injury, western blot analyses detected neurofilament 200 losses of 65% (ipsilateral) and 36% (contralateral) of levels observed in naive, uninjured rat cortices. Neurofilament 68 protein levels decreased only in the ipsilateral cortex by 35% relative to naive protein levels. Calpain inhibitor 2, administered 10 min after injury via continuous arterial infusion into the right external carotid artery for 24 h, significantly reduced neurofilament 200 losses to 17% and 3% relative to naive neurofilament 200 protein levels in the ipsilateral and contralateral cortices, respectively. Calpain inhibitor administration abolished neurofilament 68 loss in the ipsilateral cortex and was accompanied by a reduction of putative calpain-mediated neurofilament 68 breakdown products. Spectrin breakdown products mediated by calpain 1 activation were detectable in both hemispheres 24 h after traumatic brain injury and were substantially reduced in animals treated with calpain inhibitor 2 both ipsilaterally and contralaterally to the site of injury. Qualitative immunofluorescence studies of neurofilament 200 and neurofilament 68 confirmed western blot data, demonstrating morphological protection of neuronal structure throughout cortical regions of the traumatically injured brain. Morphological protection included preservation of dendritic structure and reduction of axonal retraction balls. In addition, histopathological studies employing hematoxylin and eosin staining indicated reduced extent of contusion at the injury site. These data indicate that calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons after experimental traumatic brain injury in vivo.

Experimental traumatic brain injury in rats stimulates the expression, production and activity of Alzheimer’s disease β-secretase (BACE-1)

Summary.Traumatic brain injury (TBI) is a risk factor for the development of Alzheimer’s disease (AD). After a traumatic brain injury depositions of amyloid beta (Aβ) in the brain parenchyma were found. In this study we investigated the expression pattern of β-secretase (BACE-1) in ipsi- or contralateral hippocampus and cortex following controlled cortical TBI in rats. BACE-1 mRNA levels, estimated by real time RT-PCR, were elevated 24 h post injury, and persisting up to 72 h, in the ipsi- and contralateral hippocampus and cerebral cortex as compared to the sham-treated animals (p<0.01). The TBI-induced changes in BACE-1 mRNA are due to enhanced hippocampal and cortical expression of BACE-1 mRNA in neurons and reactive astrocytes as revealed by in situ hybridization. The alterations in hippocampal BACE-1 mRNA levels are accompanied by corresponding increases in BACE-1 protein levels in ipsi- and contralateral hippocampus and ipsilateral cortex as demonstrated by Western blot analysis. In contrast, in the contralateral cortex only a weak increase of traumatically induced BACE-1 protein production was found. The activity of BACE-1 as measured by the formation of the cleavage product of amyloid beta precursor protein, transiently increased up to 48 h after injury, but returned to basal level 7 days post injury. This study demonstrates that the β-secretase is stimulated following TBI and may suggest a mechanism for the temporal increase of Aβ levels observed in patients with brain trauma.

μ-Calpain activation and calpain-mediated cytoskeletal proteolysis following traumatic brain injury

Abstract: Increasing evidence suggests that excessive activation of the calcium‐activated neutral protease μ‐calpain could play a major role in calcium‐mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of μ‐calpain after unilateral cortical impact injury in vivo, the ratio of the 76‐kDa activated isoform of μ‐calpain to its 80‐kDa precursor was measured by western blotting. This μ‐calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24–48 h after injury. We also investigated the effect of μ‐calpain activation on proteolysis of the neuronal cytoskeletal protein α‐spectrin. Immunoreactivity for α‐spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of α‐spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of α‐spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of μ‐calpain autolysis, calpain‐mediated α‐spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although μ‐calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain‐mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent μ‐calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.

Temporal Profile and Cell Subtype Distribution of Activated Caspase‐3 Following Experimental Traumatic Brain Injury

Abstract: This study investigated the temporal expression and cell subtype distribution of activated caspase‐3 following cortical impact‐induced traumatic brain injury in rats. The animals were killed and examined for protein expression of the proteolytically active subunit of caspase‐3, p18, at intervals from 6 h to 14 days after injury. In addition, we also investigated the effect of caspase‐3 activation on proteolysis of the cytoskeletal protein α‐spectrin. Increased protein levels of p18 and the caspase‐3‐specific 120‐kDa breakdown product to α‐spectrin were seen in the cortex ipsilateral to the injury site from 6 to 72 h after the trauma. Immunohistological examinations revealed increased expression of p18 in neurons, astrocytes, and oligodendrocytes from 6 to 72 h following impact injury. In contrast, no evidence of caspase‐3 activation was seen in microglia at all time points investigated. Quantitative analysis of caspase‐3‐positive cells revealed that the number of caspase‐3‐positive neurons exceeded the number of caspase‐3‐positive glia cells from 6 to 72 h after injury. Moreover, concurrent assessment of nuclear histopathology using hematoxylin identified p18‐immunopositive cells exhibiting apoptotic‐like morphological profiles in the cortex ipsilateral to the injury site. In contrast, no evidence of increased p18 expression or α‐spectrin proteolysis was seen in the ipsilateral hippocampus, contralateral cortex, or hippocampus up to 14 days after the impact. Our results are the first to demonstrate the concurrent expression of activated caspase‐3 in different CNS cells after traumatic brain injury in the rat. Our findings also suggest a contributory role of activated caspase‐3 in neuronal and glial apoptotic degeneration after experimental TBI in vivo.

Safety and efficacy of a novel intravascular cooling device to control body temperature in neurologic intensive care patients: A prospective pilot study

Objective To determine the safety and efficacy of a novel intravascular cooling device (Cool Line catheter with Cool Gard system) to control body temperature (temperature goal <37°C) in neurologic intensive care patients. Design A prospective, uncontrolled pilot study in 51 consecutive neurologic intensive care patients. Setting A neurologic intensive care unit at a tertiary care university hospital. Participants Patients were 51 neurologic intensive care patients with an intracranial disease requiring a central venous catheter due to the primary (intracranial) disease. We excluded patients under the age of 19 yrs and those with active cardiac arrhythmia, full sepsis syndrome, bleeding diathesis and infection, or bleeding at the site of the intended catheter insertion. Male to female ratio was 31:20, and the median age was 55 yrs (range, 24–85 yrs). Forty-four of 51 patients (86.3%) had an initial Glasgow Coma Scale score of 3, three patients had a Glasgow Coma Scale score of 9, one patient presented with an initial Glasgow Coma Scale score of 11, two patients had an initial Glasgow Coma Scale score of 13, and one patient had an initial Glasgow Coma Scale score of 15. The mean initial tissue injury severity score was 45.1 and the median initial tissue injury severity score 45.0 (range, 19–70). Interventions Patients were enrolled prospectively in a consecutive way. Within 12 hrs after admission, the intravascular cooling device (Cool Line catheter) was placed, the temperature probe was located within the bladder (by Foley catheter), and the Cool Gard cooling device was initiated. This Cool Gard system circulates temperature-controlled sterile saline through two small balloons mounted on the distal end of the Cool Line catheter. The patient’s blood is gently cooled as it is passed over the balloons. The Cool Gard system has been set with a target temperature of 36.5°C. The primary purpose and end point of this study was to evaluate the cooling capacity of this intravascular cooling device. Efficacy is expressed by the calculation formula of fever burden, which is defined as the fever time product (°C hours) under the fever curve. Measurements and Main Results The cooling device was in operation for a mean of 152.4 hrs. The ease of insertion was judged as easy in 42 of 51 patients; in a single patient, the catheter was malpositioned within the jugular vein, requiring early removal. The rate of infectious and noninfectious complications (nosocomial pneumonia, bacteremia, catheter-related ventriculitis, pulmonary embolism, etc.) was comparable to the rate usually observed in our neurologic intensive care patients with such severe intracranial diseases. The total fever burden within the entire study period of (on average) 152.4 hrs was 4.0°C hrs/patient, being equivalent to 0.6°C hrs/patient and day. Thirty of 51 patients showed an elevation of the body temperature (>37.9°C) within 24 hrs after termination of the cooling study. One awake patient (subarachnoid hemorrhage, Glasgow Coma Scale score 15) experienced mild to moderate shivering throughout the entire period of 7 days. The mortality rate was 23.5%. Conclusion This novel intravascular cooling device (Cool Line catheter and Cool Gard cooling device) was highly efficacious in prophylactically controlling the body temperature of neurologic intensive care patients with very severe intracranial disease (median Glasgow Coma Scale score, 3–15). Morbidity and mortality rates were consistent with the ranges reported in the literature for such neurologic intensive patients.

Expression of Fas and Fas ligand after experimental traumatic brain injury in the rat.

Apoptotic cell death plays an important role in the cascade of neuronal degeneration after traumatic brain injury (TBI), but the underlying mechanisms are not fully understood. However, increasing evidence suggests that expression of Fas and its ligand (FasL) could play a major role in mediating apoptotic cell death in acute and chronic neurologic disorders. To further investigate the temporal pattern of Fas and FasL expression after experimental TBI in the rat, male Sprague Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for Fas and FasL protein expression and for immunohistologic analysis at intervals from 15 minutes to 14 days after injury. Increased Fas and FasL immunoreactivity was seen in the cortex ipsilateral to the injury site from 15 minutes to 72 hours after the trauma, respectively. Immunohistologic investigation demonstrated a differential pattern of Fas and FasL expression in the cortex, respectively: increased Fas immunoreactivity was seen in cortical astrocytes and neurons from 15 minutes to 72 hours after the injury. In contrast, increased expression of FasL was seen in cortical neurons, astrocytes, and microglia from 15 minutes to 72 hours after impact injury. Concurrent double-labeling examinations using terminal deoxynucleotidyl tranferase-mediated deoxyuridine-biotin nick end labeling identified Fas- and FasL-immunopostive cells with high frequency in the cortex ipsilateral to the injury site. In contrast, there was no evidence of Fas- and FasL-immunopositive cells in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. Further, Fas and FasL immunoreactivity was absent in the contralateral cortex and hippocampus at all time points investigated. These results reveal induction of Fas and FasL expression in the cortex after TBI in the rat. Further, these data implicate an involvement of Fas and FasL in the pathophysiologic mechanism of apoptotic neurodegeneration after TBI. Last, these data suggest that strategies aimed to repress posttraumatic Fas- and FasL-induced apoptosis may open new perspectives for the treatment of TBI.

Cytoskeletal Derangements of Cortical Neuronal Processes Three Hours after Traumatic Brain Injury in Rats: An Immunofluorescence Study

Semiquantitative Western blot analyses have shown that traumatic brain injury (TBI) can produce significant loss of cytoskeletal proteins (neurofilament 68 [NF68], neurofilament 200 [NF200] and microtubule associated protein 2 [MAP2]) possibly by calpain-mediated proteolysis. Thus, we employed immunofluorescence (light and confocal microscopy) to study the histopathological correlates of acute neurofilament and MAP2 protein decreases observed 3 hours following unilateral cortical injury in rats. TBI induced dramatic alterations in NF68, NF200, and MAP2 immunolabeling in dendrites within and beyond contusion sites ipsilateral and contralateral to the injury site. Marked changes in immunolabeling were associated with but not exclusively restricted to regions of dark shrunken neurons labeled by hematoxylin and eosin staining, a morphopathological response to injury suggesting impending cell death. Light microscopic studies of NF200 immunofluorescence revealed a prominent fragmented appearance of apical dendrites of pyramidal neurons within layers 3 and 5, as well as a loss of fine dendritic arborization within layer 1. Confocal microscopy detected varying degrees of NF200 disassembly associated with these areas of neurofilament fragmentation. Light microscopic studies of NF68 immunofiuorescence detected subtle and less severe structural changes including smaller breaks and focal vacuolization of apical dendrites. Light microscopic immunofiuorescence of MAP2 revealed changes similar to those seen for NF200. Acute axonal alterations detected with NF68 were minimal compared to immunofluorescence changes seen in dendritic regions. Therefore, preferential dendritic cytoskeletal derangements may be an early morphological feature of experimental traumatic brain injury in vivo. In addition, these cytoskeletal derangements may not be exclusively restricted to sites of contusion and cell death

Concurrent assessment of calpain and caspase-3 activation after oxygen-glucose deprivation in primary septo-hippocampal cultures.

The contributions of calpain and caspase-3 to apoptosis and necrosis after central nervous system (CNS) trauma are relatively unexplored. No study has examined concurrent activation of calpain and caspase-3 in necrotic or apoptotic cell death after any CNS insult. Experiments used a model of oxygen–glucose deprivation (OGD) in primary septo-hippocampal cultures and assessed cell viability, occurrence of apoptotic and necrotic cell death phenotypes, and protease activation. Immunoblots using an antibody detecting calpain and caspase-3 proteolysis of α-spectrin showed greater accumulation of calpain-mediated breakdown products (BDPs) compared with caspase-3–mediated BDPs. Administration of calpain and caspase-3 inhibitors confirmed that activation of these proteases contributed to cell death, as inferred by lactate dehydrogenase release. Oxygen–glucose deprivation resulted in expression of apoptotic and necrotic cell death phenotypes, especially in neurons. Immunocytochemical studies of calpain and caspase-3 activation in apoptotic cells indicated that these proteases are almost always concurrently activated during apoptosis. These data demonstrate that calpain and caspase-3 activation is associated with expression of apoptotic cell death phenotypes after OGD, and that calpain activation, in combination with caspase-3 activation, could contribute to the expression of apoptotic cell death by assisting in the degradation of important cellular proteins.

Increased expression of apolipoprotein D following experimental traumatic brain injury.

Abstract : Increasing evidence suggests that apolipoprotein D (apoD) could play a major role in mediating neuronal degeneration and regeneration in the CNS and the PNS. To investigate further the temporal pattern of apoD expression after experimental traumatic brain injury in the rat, male Sprague‐Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for apoD mRNA and protein expression and for immunohistological analysis at intervals from 15 min to 14 days after injury. Increased apoD mRNA and protein levels were seen in the cortex and hippocampus ipsilateral to the injury site from 48 h to 14 days after the trauma. Immunohistological investigation demonstrated a differential pattern of apoD expression in the cortex and hippocampus, respectively : Increased apoD immunoreactivity in glial cells was detected from 2 to 3 days after the injury in cortex and hippocampus. In contrast, increased expression of apoD was seen in cortical and hippocampal neurons at later time points following impact injury. Concurrent histopathological examination using hematoxylin and eosin demonstrated dark, shrunken neurons in the cortex ipsilateral to the injury site. In contrast, no evidence of cell death was observed in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. No evidence of increased apoD mRNA or protein expression or neuronal pathology by hematoxylin and eosin staining was detected in the contralateral cortex and hippocampus. Our results reveal induction of apoD expression in the cortex and hippocampus following traumatic brain injury in the rat. Our data also suggest that increased apoD expression may play an important role in cortical neuronal degeneration after brain injury in vivo. However, increased expression of apoD in the hippocampus may not necessarily be indicative of neuronal death.