Yngve Olsson

Regional changes in interstitial K+ and Ca2+ levels following cortical compression contusion trauma in rats

Brain trauma is associated with acute functional impairment and neuronal injury. At present, it is unclear to what extent disturbances in ion homeostasis are involved in these changes. We used ion-selective microelectrodes to register interstitial potassium ([K+]e) and calcium ([Ca2+]e) concentrations in the brain cortex following cerebral compression contusion in the rat. The trauma was produced by dropping a 21 g weight from a height of 35 cm onto a piston that compressed the cortex 1.5 mm. Ion measurements were made in two different locations of the contused region: in the perimeter, i.e., the shear stress zone (region A), and in the center (region B). The trauma resulted in an immediate increase in [K+]e from a control level of 3 mM to a level >60 mM in both regions, and a concomitant negative shift in DC potential. In both regions, there was a simultaneous, dramatic decrease in [Ca2+]e from a baseline of 1.1 mM to 0.3–0.1 mM. Interstitial [K+] and the DC potential normalized within 3 min after trauma. In region B, [Ca2+]e recovered to near control levels within 5 min after ictus. In region A, however, recovery of [Ca2+]e was significantly slower, with a return to near baseline values within 50 min after trauma. The prolonged lowering of [Ca2+]e in region A was associated with an inability to propagate cortical spreading depression, suggesting a profound functional disturbance. Histologic evaluation 72 h after trauma revealed that neuronal injury was confined exclusively to region A. The results indicate that compression contusion trauma produces a transient membrane depolarization associated with a pronounced cellular release of K+ and a massive Ca2+ entry into the intracellular compartment. We suggest that the acute functional impairment and the subsequent neuronal injury in region A is caused by the prolonged disturbance of cellular calcium homeostasis mediated by leaky membranes exposed to shear stress.

Changes in microtubule-associated protein 2 and amyloid precursor protein immunoreactivity following traumatic brain injury in rat: influence of MK-801 treatment

We investigated by immunohistochemistry dendritic and axonal changes occurring in the rat brain after mild focal cortical trauma produced by the weight drop technique. One and 3 days after injury, nerve cell bodies and dendrites in the perimeter of the impact site displayed decreased microtubule-associated protein 2 (MAP2) immunoreactivity. Some dendrites in the immediate adjacent region were more intensely stained and distorted. The dentate hilar region of the hippocampus showed a reduction of immunoreactive nerve cell bodies and dendrites. Twenty-one days after injury the strongly stained cortical dendrites and the reduction of immunoreactivity in the hippocampus remained, whereas the reduced staining in the perimeter of the lesion had normalised. These results indicate that there is a long-lasting disturbed dendritic organisation implicating impaired neurotransmission after this type of mild brain trauma. beta-Amyloid precursor protein (APP) immunohistochemistry revealed numerous stained axons in the ipsilateral subcortical white matter and thalamus indicating local and remote axonal injuries with disturbed axonal transport. Twenty-one days after injury, numerous small immunostained profiles appeared in the neuropil of the cortical impact site and in the ipsilateral thalamus. The axonal changes indicate disturbed connectivity between the site of the impact and other brain regions, chiefly the thalamus. The presence of beta-amyloid was investigated 21 days after trauma. There were no signs of beta-amyloid depositions in the brain after injury. Finally, we tested if the non-competitive NMDA receptor antagonist dizocilpine maleate (MK-801) could influence the observed MAP2 and APP changes. Pretreatment with this compound did not affect the early MAP2 and APP alterations. Instead, an increased expression of the APP antigen in the thalamus was observed 21 days after trauma in the MK-801-treated animals. The cause of this phenomenon is not known but may be related to a delayed neurotoxic action of MK-801 treatment.

Traumatic brain injury in rat produces changes of β-amyloid precursor protein immunoreactivity

beta-Amyloid precursor protein immunoreactivity (APP) was studied after a mild compression contusion trauma to rat parietal cortex. Neurones in the periphery of the cortical lesion, i.e. tissue subjected to shear stress, showed markedly reduced immunoreactivity 1 and 3 days after injury. Numerous axons in the ipsilateral subcortical white matter and thalamus became immunoreactive. At 21 days, small rounded profiles appeared in the neuropil of the damaged cortex and in the thalamus. Thus, traumatic brain injury appears to induce several types of APP changes. The accumulation in neuronal processes is probably caused by disturbed axonal transport induced by trauma. Since APP is assumed to be excitoprotective, modulating intracellular Ca2+ responses, the decreased immunoreactivity noticed in the periphery of the lesion may render the neurones in this region more vulnerable to secondary injury mechanisms.

A metabolic threshold of irreversible ischemia demonstrated by PET in a middle cerebral artery occlusion–reperfusion primate model*

Objective– to evaluate the predictive value of measurements of regional cerebral blood flow (CBF), oxygen metabolism (CMRO2) and oxygen extraction ratio (OER) for assessment of the fate of ischemic brain tissue. Materials and methods– Sequential PET measurements were performed during middle cerebral artery occlusion (MCAO; 2 h) and 12–24 h (mean 18 h) of reperfusion in a primate model (Macaca mulatta, n=8). A penumbra region was delineated on the MCAO PET image (OER >125% and CMRO2≥45% of the values observed in the contralateral hemisphere, respectively) and an infarction region was delineated on the last PET image (CMRO2<45% of the values observed in the contralateral hemisphere). The penumbra regions delineated during MCAO and the infarction regions delineated at the final PET, were copied on to the images from all other PET sessions for measurements of CBF, CMRO2 and OER. Ratios were calculated by dividing the mean values obtained by the values of the corresponding contralateral region. Results– Histopathology verified the adequacy of the criteria applied in the last PET for delineation of the infarction region. The penumbra region and infarction region were separated in all cases, except in two cases where a minimal overlap was seen. CBF and OER showed considerable variation over time and there was no consistent difference between the penumbra and infarction regions. CMRO2 showed a more stable pattern and the difference between penumbra and infarction regions was maintained from the time of MCAO throughout the entire reperfusion phase. With CMRO2 as predictor, all 50 observations could be correctly predicted as penumbra or infarction when using an optimal threshold ratio value estimated to be in the interval of 61% to 69% of the corresponding contralateral region. CBF and OER proved to have low power as predictors. Conclusions– The results indicate that CMRO2 is the best predictor of reversible or irreversible brain damage and the critical metabolic threshold level appears to be a reduction of oxygen metabolism to between 61% and 69% of the corresponding contralateral region.

alpha-Phenyl-tert-N-butyl nitrone (PBN) improves functional and morphological outcome after cortical contusion injury in the rat.

Summaryu2003α-Phenyl-tert-N-butyl nitrone (PBN), a potent reactive oxygen species (ROS) scavenger, has shown robust neuroprotective properties in several models of acute brain injury, although not previously evaluated in traumatic brain injury (TBI). In this study, we assessed the potential efficacy of PBN in a weight drop model producing a controlled cortical contusion. Sham operation, mild or severe injury was induced in intubated and ventilated rats and functional and morphological outcome was used as end-points at two weeks post-injury. In the trauma groups, saline or PBN (30 mg/kg) was injected as an intravenous bolus 30 minutes prior to injury. At day 11–15 post-injury, cognitive disturbance was assessed using the Morris Water Maze (MWM) and estimation of lesion volume and hemispheric loss of tissue was made. No change in MWM performance were found in either of the mildly traumatized groups as compared to uninjured controls. In contrast, a significant decrease in total mean latency and increase in path length in the severely traumatized rats were found. PBN-treatment significantly improved MWM performance as compared to saline treatment at the severe injury level (p<0.05). The mild injury level caused a discrete atrophy of the ipsilateral cortex with no effect of PBN treatment. The severe injury caused a substantial loss of ipsilateral hemispheric tissue and a large cortical cavitation. PBN pre-treatment significantly reduced the lesion volume and reduced hemispheric loss of tissue at this injury level (p<0.05).u2003Our results support the involvement of ROS in the injury process contributing to the tissue loss and cognitive disturbance after TBI. The potential clinical utility of PBN will have to be assessed using a post-injury dosing regime.

Extracellular amino acid levels measured with intracerebral microdialysis in the model of posttraumatic epilepsy induced by intracortical iron injection.

An iron induced model of posttraumatic chronic focal epilepsy in rats was studied with respect to extracellular amino acids, electrophysiology, and morphology, approx. 6 months after intracortical injection of ferrous chloride. Twenty-six of the twenty-eight (93%) rats developed spontaneous epileptiform EEG-activity and electrical cortical stimulation done in eight animals evoked seizure activity in five animals (62.5%). Epileptic brain tissue displayed significantly higher extracellular interictal levels of aspartate (ASP), compared to normal brain, measured with intracerebral microdialysis. The interictal levels of serine (SER) were significantly higher at the lesion side compared to the contralateral cortex in epileptic animals. Spontaneous elevations of ASP and glutamate (GLU) levels up to 8 times the basal level were found in 4/5 (80%). There was no consistent amino acid pattern following the electrically induced seizures, but in association with more intense seizure activity ASP and GLU were elevated. Histopathologically, the necrotic lesions in the cortex contained small vessels and iron pigment loaded astrocytes. Scattered eosinophilic neurons were found in the hippocampus, bilaterally in 37% of the animals. The results show that a focal epileptiform activity developed in a high percentage of animals that received an intracortical iron injection. The observed amino acid changes in epileptic animals may be involved in the development of seizures in this model of posttraumatic epilepsy.

Behavioural and Morphological Outcome of Mild Cortical Contusion Trauma of the Rat Brain: Influence of NMDA-Receptor Blockade

Summaryu2003The authors studied the effect of a mild cortical contusion to the rat brain on behavioural and morphological outcome and the influence of NMDA-receptor blockade (MK-801, 0.5 mg/kg i.v. 30 min prior to trauma). Spontaneous motor activity was assessed 16–18 days post trauma. Saline treated traumatised rats showed a significant (p<0.01) hyperactive behaviour compared to animals without injury. MK-801 treated rats performed significantly better than the saline treated animals (p<0.05). For histopathological evaluation hippocampal hilar neurons were counted, cortical thickness under the impact was measured and microtubule-associated protein 2 (MAP2) immunoreactivity in the dentate hilus was quantified 1, 3 and 21 days post trauma. In traumatised rats scattered loss of nerve cells, oedema and minute haemorrhages were present at the site of the impact one and three days after injury. At day 21 there was a significant reduction of cortical thickness at the site of impact. One day after trauma there was a bilateral, significant loss of neurons and MAP2 immunostaining in the dentate hilus of the hippocampus. MK-801 pretreated rats showed similar morphological changes. The disturbed spontaneous motor behaviour may be caused by hippocampal damage and a reduction of somatosensory cortical neurons. NMDA-receptor blockade improved the outcome assessed by the functional tests but failed to influence the morphological changes, suggesting that this behavioural test is a more sensitive indicator of outcome after mild traumatic brain injury (TBI).

Correlation of Hippocampal Morphological Changes and Morris Water Maze Performance after Cortical Contusion Injury in Rats

OBJECTIVE:The hippocampus is essential to the processing and formation of memory. This study analyzed the relationship among memory dysfunction as revealed by Morris water maze (MWM) trial, cortical lesion volume, and regional hippocampal morphological changes after controlled cortical contusion (CCC). We also analyzed the influence of pretreatment with the nitrone radical scavenger &agr;-phenyl-N-tert-butyl-nitrone (PBN). METHODS:Rats were subjected to CCC. We used two levels of CCC (mild, 1.5 mm and severe, 2.5 mm) and pretreated some severely injured animals with PBN. The animals were killed 15 days postinjury. We evaluated morphological changes to the hippocampus semiquantitatively by scoring sections immunohistochemically stained for microtubule-associated protein 2 with a four-point scale for the cornu ammonis (CA) 1, CA2, CA3, and hilus of the dentate gyrus (HDG). The cortical lesion volume was quantified. RESULTS:Rats subjected to severe, but not mild, CCC demonstrated impaired spatial learning ability in the MWM, but this impairment was attenuated with pretreatment with the radical scavenger PBN. We documented bilateral morphological changes in CA1, CA3, and HDG and an ipsilateral neocortical cavitation in severely injured rats. PBN treatment attenuated (P < 0.05) the morphological characteristics of abnormality in the ipsilateral CA1, CA2, HDG, and the contralateral HDG and reduced the cortical lesion volume. Mild injury led to minor ipsilateral hippocampal and cortical damage but no MWM deficiency. Hippocampal morphological scores and total mean latencies in the MWM task were strongly correlated (r = 0.69; P < 0.001). The correlation between the cortical lesion volume and MWM latency was weaker (r = 0.48; P = 0.02). CONCLUSION:Severe CCC causes bilateral morphological changes in the hippocampus and ipsilateral neocortical cavitation, which correlate to impairment in a spatial learning task (MWM). PBN protected the structure of the CA2 ipsilaterally and HDG bilaterally and reduced the cortical lesion volume, correlating to improved functional outcome.

Effect of the 21-aminosteroid U74006F and methylprednisolone on motor function recovery and oedema after spinal cord compression in rats

The effect of the 21‐aminosteroid U74006F and methylprednisolone (MP) on motor function and oedema were investigated after spinal cord compression in rats. Each compound was administered i.v. as a single dose 60 min after injury. The hind limb motor function was assessed using the inclined plane technique and expressed as the capacity angle. The water content was calculated as the percent wet weight of the total weight. Prior to compression the capacity angle was close to 62–64°. One day after compression the motor function was reduced significantly in all rats. However, the capacity angle was significantly higher after treatment with U74006F or MP than with vehicle, i.e. 50°± 4, 45°± 5, and 32°± 3, respectively. This improved functional recovery persisted during the initial nine days. After compression of the spinal cord the water content increased to a maximum on day 4 in all groups. The water content was not significantly different in any of the groups except on day one and nine when it was less in groups treated with U74006F. In conclusion, a single i. v. injection of U74006 or MP given 60 min after compression of the spinal cord improved motor function without effecting oedema expressed as water content.

Clomethiazole (ZENDRA, CMZ) improves hind limb motor function and reduces neuronal damage after severe spinal cord injury in rat.

Abstract Clomethiazole (CMZ) has a neuroprotective effect in experimental focal and global forebrain ischemia. This neuroprotective effect may depend on its ability to enhance GABA receptor activity. We have studied the effect of pretreatment with CMZ on motor function recovery and nerve cell damage after spinal cord injury (SCI). Rats were randomized and 30 min before SCI they received a single intraperitoneal dose of CMZ (150 mg/kg) or saline. The spinal cord was injured with a 50 g (4.5 g/mm2) load, applied over the exposed dura, through a curved rectangular plate (2.2 × 5.0 mm) for 5 min at T8–9. The animals became paraplegic 1 day after injury. The rats were evaluated for recovery of hind limb motor function. All animals recovered to some extent over the observation period of 12 weeks. However, hind limb motor function was significantly better in the animals pretreated with CMZ. At 12 weeks the rats were killed and perfused/fixed for morphological investigations. Microtubule-associated protein 2 (MAP2) immunostaining was used to stain neurons and dendrites and Luxol-fast blue to stain myelinated tracts of the white matter. The injured segment of the spinal cord showed severe atrophy, distortion, cavitation and necrosis of grey and white matter. Compared to uninjured controls the transverse sectional area was reduced to 32.7 ± 4% in untreated animals but only to 38.5% ± 4.1 in CMZ-treated animals. MAP2 staining showed that, compared to uninjured controls, grey matter was reduced to 7.4 ± 2.7% in saline-treated injured animals and to 22.7 ± 5.4% in CMZ-treated rats. Our results thus show that in this model CMZ improves hind limb motor function and attenuates the morphological damage to the spinal cord.