Biomedical subjects

A Kampfl

Publications and source records attributed to A Kampfl.

At least 37 records · Page 2Linked to original sources

PubMed1998

Subcellular localization and duration of mu-calpain and m-calpain activity after traumatic brain injury in the rat: a casein zymography study.

Casein zymographic assays were performed to identify changes in mu-calpain and m-calpain activity in naive, sham-injured, and injured rat cortex at 15 minutes, 3 hours, 6 hours, and 24 hours after unilateral cortical impact brain injury. Cortical samples ipsilateral and contralateral to the site of injury were separated into cytosolic and total membrane fractions. Marked increases in mu-calpain activity in cytosolic fractions in the ipsilateral cortex occurred as early as 15 minutes, became maximal at 6 hours, and decreased at 24 hours to levels observed at 15 minutes after injury. A similar temporal profile of cytosolic mu-calpain activity in the contralateral cortex was observed, although the increases in the contralateral cortex were substantially lower than those in the ipsilateral cortex. Differences were also noted between cytosolic and total membrane fractions. The detection of a shift in mu-calpain activity to the total membrane fraction first occurred at 3 hours after traumatic brain injury and became maximal at 24 hours after traumatic brain injury. This shift in mu-calpain activity between the two fractions could be due to the redistribution of mu-calpain from the cytosol to the membrane. m-Calpain activity was detected only in cytosolic fractions. m-Calpain activity in cytosolic fractions did not differ significantly between ipsilateral and contralateral cortices, and increased in both cortices from 15 minutes to 6 hours after injury. Relative magnitudes of m-calpain versus mu-calpain activity in cytosolic fractions differed at different time points after injury. These studies suggest that traumatic brain injury can activate both calpain isoforms and that calpain activity is not restricted to sites of focal contusion and cell death at the site of impact injury but may represent a more global response to injury.

Animals↗

PubMed1998

The persistent vegetative state after closed head injury: clinical and magnetic resonance imaging findings in 42 patients.

OBJECT: In this retrospective study, the authors analyzed the frequency, anatomical distribution, and appearance of traumatic brain lesions in 42 patients in a posttraumatic persistent vegetative state. METHODS: Cerebral magnetic resonance (MR) imaging was used to detect the number of lesions, which ranged from as few as five to as many as 19, with a mean of 11 lesions. In all 42 cases there was evidence on MR imaging of diffuse axonal injury, and injury to the corpus callosum was detected in all patients. The second most common area of diffuse axonal injury involved the dorsolateral aspect of the rostral brainstem (74% of patients). In addition, 65% of these patients exhibited white matter injury in the corona radiata and the frontal and temporal lobes. Lesions to the basal ganglia or thalamus were seen in 52% and 40% of patients, respectively. Magnetic resonance imaging showed some evidence of cortical contusion in 48% of patients in this study; the frontal and temporal lobes were most frequently involved. Injury to the parahippocampal gyrus was detected in 45% of patients; in this subgroup there was an 80% incidence of contralateral peduncular lesions in the midbrain. The most common pattern of injury (74% in this series) was the combination of focal lesions of the corpus callosum and the dorsolateral brainstem. In patients with no evidence of diffuse axonal injury in the upper brainstem (26% in this series), callosal lesions were most often associated with basal ganglia lesions. Lesions of the corona radiata and lobar white matter were equally distributed in patients with or without dorsolateral brainstem injury. Moreover, cortical contusions and thalamic, parahippocampal, and cerebral peduncular lesions were also similarly distributed in both groups. CONCLUSIONS: The data indicate that diffuse axonal injury may be the major form of primary brain damage in the posttraumatic persistent vegetative state. In addition, the authors demonstrated in this study that MR imaging, in conjunction with a precise clinical correlation, may provide useful supportive information for the accurate diagnosis of a persistent vegetative state after traumatic brain injury.

Adolescent↗

PubMed1998

Potential contribution of proteases to neuronal damage.

Calpain was first discovered 30 years ago. Two major isoforms were subsequently isolated and purified. The presence of an endogenous protein inhibitor, calpastatin, was later discovered. Calpain activity is tightly regulated by Ca(2+). At physiological levels of Ca(2+), the role of calpain remains poorly understood, but is believed to be involved in mitosis and muscle cell differentiation. Calpain has also been implicated in various membrane fusion events through remodeling of the cytoskeletal network. Calpain activation has been shown to be increased during normal aging and in muscular dystrophy, cataract, arthritis and Alzheimer's disease, and in acute traumas such as traumatic brain injury (TBI), spinal cord injury and cerebral and cardiac ischemia. Early work on calpain inhibitors was limited to protein inhibitors and other nonselective enzyme inhibitors. Peptidyl aldehydes such as leupeptin and antipain are also among the earliest reported calpain inactivators. Irreversible inhibitors such as the E64 family have also been studied, and peptidyl halomethanes and diazomethanes have long been used as protease inhibitors. A variety of calpain inhibitors are under development. From a therapeutic perspective, calpain inhibitors may have several advantages over other more conventional targets such as ion channel blockers and glumate antagonists, since calpain proteolysis represents a later component of a pathway mediating cell death initiated by excitotoxicity and elevated Ca(2+) levels. Although the potential clinical utility of calpain inhibitors seems well established, a number of important considerations remain to be addressed. The role of other proteolytic cascades contributing to neuronal cell damage following TBI must also be considered.

Journal Article↗

PubMed1997

Near infrared spectroscopy (NIRS) in patients with severe brain injury and elevated intracranial pressure. A pilot study.

Near infrared spectroscopy (NIRS) was used to asses changes in regional cerebral oxygen saturation (rSO2) in 8 head injured patients with an intracranial pressure (ICP) higher or lower than 25 mmHg (n = 4 for each group). NIRS values in the high ICP group (> 25 mmHg) were significantly lower than in the low ICP group (< 25 mmHg). In contrast, arterial pO2, pCO2, peripheral oxygen saturation and transcranial Doppler sonography (TCD) values were similar in both groups. To further investigate changes in rSO2 to changes in peripheral oxygen saturation and arterial pO2, patients of both groups underwent an artificial hyperoxygenation (50% O2) period of 3 minutes. Both groups revealed similar values in peripheral oxygen saturation, arterial pO2, and TCD velocities at the end of the hyperoxygenation period. However, rSO2 values in patients with an ICP > 25 mmHg were significant lower than in patients with an ICP < 25 mmHg after the hyperoxygenation period. In addition, patients with an ICP < 25 mmHg revealed a significant increase in rSO2 values at the end of the hyperoxygenation period, not detectable in patients with an ICP > 25 mmHg. Our results suggest that NIRS may be an additional diagnostic tool in the non-invasive evaluation of impaired cerebral microcirculation in patients with increased intracranial pressure.

Adult↗

PubMed1997

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.

Animals↗

PubMed1997

Cerebrospinal fluid (CSF) pharmacokinetics of intraventricular vancomycin in patients with staphylococcal ventriculitis associated with external CSF drainage.

We studied the efficacy and pharmacokinetics of intraventricularly administered vancomycin in three patients with shunt-associated staphylococcal ventriculitis. We instilled 10 mg of the drug intraventricularly every 24 hours. Cerebrospinal fluid (CSF) levels were measured 1 hour after instillation and then every 2 hours. Peak vancomycin levels reached a mean of 292.9 microg/mL. The mean trough levels, measured immediately before readministration of vancomycin, were 7.6 microg/mL; this level has proved to be sufficient for maintaining the necessary steady-state serum concentration of vancomycin. All three patients were cured clinically and bacteriologically, and CSF parameters returned to normal within 5-13 days. No side effects were observed. Our results suggest that intraventricularly administered vancomycin is a valuable therapeutic strategy for treating shunt-associated staphylococcal ventriculitis. In addition, we provide evidence that 10 mg of vancomycin, administered intraventricularly every 24 hours, allows maintenance of therapeutic drug levels in the CSF for at least 24 hours.

Brain Injuries↗

PubMed1997

Mechanisms of calpain proteolysis following traumatic brain injury: implications for pathology and therapy: implications for pathology and therapy: a review and update.

Much recent research has focused on the pathological significance of calcium accumulation in the central nervous system (CNS) following cerebral ischemia, spinal cord injury (SCI), and traumatic brain injury (TBI). Disturbances in neuronal calcium homeostasis may result in the activation of several calcium-sensitive enzymes, including lipases, kinases, phosphatases, and proteases. One potential pathogenic event in a number of acute CNS insults, including TBI, is the activation of the calpains, calcium-activated intracellular proteases. This article reviews new evidence indicating that overactivation of calpains plays a major role in the neurodegenerative cascade following TBI in vivo. Further, this article presents an overview from in vivo and in vitro models of CNS injuries suggesting that administration of calpain inhibitors during the initial 24-h period following injury can attenuate injury-induced derangements of neuronal structure and function. Lastly, this review addresses the potential contribution of other proteases to neuronal damage following TBI.

Animals↗

PubMed1997

Immunohistochemical study of calpain-mediated breakdown products to alpha-spectrin following controlled cortical impact injury in the rat.

This study examined the effect of unilateral controlled cortical impact on the appearance of calpain-mediated alpha-spectrin breakdown products (BDPs) in the rat cortex and hippocampus at various times following injury. Coronal sections were taken from animals at 15 min, 1 h, 3 h, 6 h, and 24 h after injury and immunolabeled with an antibody that recognizes calpain-mediated BDPs to alpha-spectrin (Roberts-Lewis et al., 1994). Sections from a separate group of rats were also taken at the same times and stained with hematoxylin and eosin. Analyses of early time points (15 min, 1 h, 3 h, and 6 h following injury) revealed alpha-spectrin BDPs in structurally intact neuronal soma and dendrites in cortex ipsilateral to site of injury that was not present in tissue from sham-injured control rats. By 24 h after injury labeling was not restricted to clearly defined neuronal structures in ipsilateral cortex, although there was an increased extent of diffuse labeling. BDPs to alpha-spectrin in axons were not detected until 24 h after injury, in contrast to the more rapid accumulation of BDPs observed in neuronal soma and dendrites. The presence of BDPs to alpha-spectrin in the cortex at the site of impact, and in the rostral and contralateral cortex, coincided with morphopathology detected by hematoxylin and eosin. alpha-Spectrin BDPs were also observed in the hippocampus ipsilateral to the injury in the absence of overt cell death. This investigation provides further evidence that calpain is activated after controlled cortical impact and could contribute to necrosis at the site of injury. The appearance of calpain-mediated BDPs at sites distal to the contusion site and in the hippocampus also suggests that calpain activation may precede and/or occur in the absence of extensive morphopathological changes.

Animals↗

PubMed1997

Impaired microcirculation and tissue oxygenation in human cerebral malaria: a single photon emission computed tomography and near-infrared spectroscopy study.

Serial single photon emission computed tomography (SPECT), near-infrared spectroscopy (NIRS), and transcranial doppler (TCD) sonography examinations were performed to investigate changes of cerebral perfusion and tissue oxygenation in a patient with complicated cerebral malaria that have been acquired in Nigeria. On admission to the Neurologic Intensive Care Unit in Innsbruck, Austria, SPECT and NIRS revealed focal right hemispheric hypoperfusion and decreased oxygen saturation, respectively, correlating exactly to the patient's right hemispheric localizing signs. In contrast, TCD examinations of the basal cerebral vessels revealed normal flow patterns. The patient showed an initial Plasmodium falciparum parasitemia rate of 30% and was cured by intravenous quinine and oral mefloquine therapy. He was discharged without neurologic symptoms. Follow-up SPECT and NIRS examinations revealed regular cerebral perfusion and oxygenation patterns in both cortical hemispheres. In summary, the presented findings provide first evidence that noninvasive SPECT and NIRS may be important diagnostic tools in the evaluation of impaired cerebral microcirculation in patients with P. falciparum malaria.

Adult↗

PubMed1996

Cationic lipid-mediated NGF gene transfection increases neurofilament phosphorylation.

We examined the effect of cationic lipid-mediated gene transfection of nerve growth factor (NGF) in primary septo-hippocampal cell cultures. Rat NGF cDNA was subcloned into a pUC19-based plasmid containing a CMV promoter. Two days after NGF gene transfection in primary cell cultures, ELISA confirmed increases in NGF protein secretion from transfected cells. To study the biological effect of cationic lipid-mediated NGF gene transfection, we analyzed the amount of neurofilament protein from NGF-transfected cell cultures. Western blot and immunohistochemical analyses detected significant increases in the phosphorylated form of neurofilament proteins in the cultures after cationic lipid-mediated NGF cDNA transfection. Cationic lipid-mediated NGF cDNA transfection did rot cause significant changes in the total amount of neurofilament protein. Our studies suggest that cationic lipid-mediated NGF gene transfection can increase neurofilament phosphorylation but not total neurofilament protein.

Animals↗

PubMed1996

mu-calpain activation and calpain-mediated cytoskeletal proteolysis following traumatic brain injury.

Increasing evidence suggests that excessive activation of the calcium-activated neutral protease mu-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 mu-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated isoform of mu-calpain to its 80-kDa precursor was measured by western blotting. This mu-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 mu-calpain activation on proteolysis of the neuronal cytoskeletal protein alpha-spectrin. Immunoreactivity for alpha-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of alpha-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 alpha-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 mu-calpain autolysis, calpain-mediated alpha-spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although mu-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 mu-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.

Analysis of Variance↗

PubMed1996

Diminished microtubule-associated protein 2 (MAP2) immunoreactivity following cortical impact brain injury.

This study employed Western blotting and qualitative immunohistochemistry to analyze the effects of cortical impact traumatic brain injury (TBI) on acute changes in MAP2 immunoreactivity in the rat cortex. We employed a lateral cortical impact injury device to induce severe TBI, which is associated with focal cortical contusion and neuronal death at the impact site. Three hours following TBI, Western blotting detected substantial MAP2 loss only in the cortex ipsilateral to the site of injury. Light microscopic studies of MAP2 revealed a prominent loss of MAP2 immunofluorescence in apical dendrites of pyramidal neurons within layers 3 and 5, as well as a loss of fine dendritic arborization within layer 1. These changes in MAP2 immunolabeling were associated with, but not exclusively restricted to, the presence of dark shrunken neurons labeled by hematoxylin and eosin staining, suggesting impending cell death. Alterations in MAP2 immunofluorescence were found both within and beyond areas of focal contusion and necrosis in the ipsilateral cortex. Thus, traumatic brain injury in rats can produce rapid and significant dendritic pathology within sites of contusion. However, immunohistochemical changes in MAP2 labeling outside of contused regions suggests that TBI-induced dendritic damage may not be exclusively associated with acute cell death.

Animals↗

PubMed1996

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 immunofluorescence detected subtle and less severe structural changes including smaller breaks and focal vacuolization of apical dendrites. Light microscopic immunofluorescence 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.

Animals↗

PubMed1996

Calpain inhibitors protect against depolarization-induced neurofilament protein loss of septo-hippocampal neurons in culture.

We examined the effect of a 6 min depolarization with 60 mM KCl and 1.8, 2.8 or 5.8 mM extracellular CaCl2 on neurofilament proteins of high (NF-H), medium (NF-M) and low (NF-L) molecular weight in primary septohippocampal cultures. One day after depolarization, Western blot analyses revealed losses of all three neurofilament proteins. Increasing the extracellular calcium concentration from 1.8 to 5.8 mM CaCl2 in the presence of 60 mM KCl produced increased losses of all three neurofilament proteins to approximately 80% of control values in the absence of cell death. Calcium-dependent losses of the neurofilament proteins correlated with calcium-dependent increases in calpain 1-mediated breakdown products of alpha-spectrin. Calpain inhibitors 1 and 2, applied immediately after depolarization and made available to cultures for 24 h, reduced losses of all three neurofilament proteins to approximately 14% of control values. The protective effects of calpain inhibitors 1 and 2 were influenced by different levels of extracellular calcium. Qualitative immunohistochemical evaluations confirmed semiquantitative Western blot data on neurofilament loss and protection by calpain inhibitors 1 and 2. We propose that brief depolarization causes loss of neurofilament proteins, possibly due to calpain activation. Thus, calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons.

Animals↗

PubMed1995

Brief potassium depolarization decreases levels of neurofilament proteins in CNS culture.

Little is known about the effects of brief potassium depolarization that occurs concurrently with transient ischemia, epilepsy and head trauma. To investigate the effect of short-term depolarization on light (NF-L), middle (NF-M), and heavy (NF-H) neurofilament proteins and determine the role played by calcium in that effect, mixed septo-hippocampal cultures were exposed to 60 mM K+ for 6 min, in the presence of 0 to 11.8 mM Ca2+. Twenty-four hours later, neurofilament immunoreactivity in Western blots of depolarized cultures was decreased to 60% or less of control levels. Decreases were Ca2+-dependent, not due to cell loss, and affected both phosphorylated and nonphosphorylated proteins. The phosphorylation state of NF-M and NF-H influenced the degree of loss observed. Changes in the pattern of immunolabelling of neuritic processes were also associated with depolarization. Thus, brief potassium depolarization may contribute to cytoskeletal disruption following brain injury.

Animals↗

PubMed1995

Time course of neurofilament protein loss following depolarization-induced injury in CNS culture.

In septo-hippocampal cell cultures, brief potassium depolarization produces calcium-dependent decreases in neurofilament proteins and loss of fine neuritic processes within 24 h. It is not known whether neurons later exhibit delayed degeneration and die, live with enduring neurofilament loss, or restore neurofilament protein levels. Therefore, we exposed septohippocampal cultures to 6 min potassium depolarization (60 mM) with 2.8-11.8 mM extracellular Ca2+ and evaluated immunoreactivity for low, medium and heavy neurofilament proteins, neuronal number, and neuronal morphology for 10 days. Neuronal number remained unchanged; neurofilament protein levels recovered to between 31% and 99% of control levels, and fine neuritic processes reappeared.

Animals↗

PubMed1995

Calpain inhibitors reduce depolarization induced loss of tau protein in primary septo-hippocampal cultures.

We studied the effects of a 6-min potassium depolarization injury produced by 60 mM KCl and 1.8 mM or 5.8 mM extracellular CaCl2 on tau protein levels in primary rat septo-hippocampal cultures. One day after injury, Western blot analyses revealed a calcium dependent loss of tau protein of approximately 50% of control values. Loss of tau protein was associated with calpain 1 mediated breakdown products to alpha-spectrin. Calpain inhibitors 1 and 2, applied immediately after depolarization injury and available to cultures for 24 h reduced depolarization induced degradation of tau protein to approximately 35% or 25% of control values, respectively. We propose that brief potassium depolarization causes degradation of tau protein, possibly due to calpain activation. Thus, calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons.

Animals↗

PubMed1995

Rescue of injury-induced neurofilament loss by BDNF gene transfection in primary septo-hippocampal cell cultures.

We employed primary septo-hippocampal cell cultures to determine the ability of liposome-mediated BDNF gene transfection to facilitate recovery of neurofilament loss caused by depolarization injury. After BDNF gene transfection in uninjured cultures, RT-PCR and immunohistochemical staining confirmed increases in BDNF mRNA and protein in transfected cells. Three days after depolarization injury, Western blot and immunohistochemical analyses detected significant loss of neurofilament proteins in non-transfected cultures, while BDNF transfection produced marked increases in neurofilament proteins following either pre-injury transfection or transfection 24 h following injury. Immunohistochemical studies also detected enhanced immunolabeling of BDNF and total neurofilament protein (phosphorylated and non-phosphorylated) in injured neurons following BDNF transfection or administration of exogenous BDNF protein, compared to untransfected, injured controls.

Animals↗