Traumatic brain injury enhances the formation of heterotopic ossification around the hip: an animal model study.

INTRODUCTION: The incidence of heterotopic ossification (HO) is at its highest when trauma of the hip or pelvis concurs with traumatic brain injury (TBI). The pathogenic mechanisms underlying the neurogenic enhancement of the formation of HO remain, however, poorly understood. Hence, the goal of the present study was to develop a novel small animal model that combines hip and brain trauma that can prove the enhancement of HO around the hip after TBI. MATERIALS AND METHODS: Forty male Wistar rats were divided into four groups, to undergo hip surgery alone (group 1), hip surgery + moderate TBI (group 2), hip surgery + severe TBI (group 3) and only severe TBI (group 4). The femoral canal was reamed up to 2 mm and a muscle lesion was made to simulate hip surgery. An established controlled cortical impact model was used to create a TBI. Twelve weeks after surgery, the hip with the proximal half of the femur and the pelvic bone was removed and subjected to micro-computed tomography (µCT) analysis. A quantitative analysis using a modified Brooker score as well as a quantitative analysis using a bone-to-tissue ratio was used. RESULTS: No HO could be found in all the ten animals that did not undergo hip surgery (group 4). In the animals that did undergo surgery to the hip, no HO was found in only one animal (group 1). All the other animals developed HO. In this study, significantly more HO was found in animals that underwent an additional severe TBI. CONCLUSION: The newly developed rat model, with a combined hip and brain trauma, showed an enhancement of the HO formation around the hip after severe TBI.

Thrombin contributes to the injury development and neurological deficit after acute subdural hemorrhage in rats only in collaboration with additional blood-derived factors.

BACKGROUND: Acute subdural hemorrhage (ASDH) is a severe consequence of traumatic brain injury. The occurrence of subdural blood increases the lethality of these patients independent of the amount of blood or elevated intracranial pressure. Thrombin is one of the potential harmful blood components. Possible harmful effects of thrombin are mediated via the Protease-activated-receptor-1 (PAR1) and thus, translating the acute Thrombin release after ASDH into cell loss. The objectives of the present study were twofold, namely to examine (1) the impact of direct thrombin inhibition in the acute phase after hemorrhage on the long-term histological and functional deficits and (2) the early inhibition of PAR1 activation by thrombin with the selective antagonist SCH79797 on lesion volume at 14 days after ASDH. The effects of thrombin on the lesion size were investigated in two separate experiments via (1) direct thrombin inhibition in the subdural infused blood (Argatroban 600 µg) as well as by (2) intraventricular injection of the PAR-1 antagonist SCH79797 (1 µg or 5 µg). Lesion volume and behavior deficits using a neurological deficit score and a motor function test (beam balance test) were analyzed as outcome parameters at 14 days after injury. RESULTS: 59 Male Sprague-Dawley rats received a subdural infusion of 300 µl autologous blood or sham operation. Lesion volume at 14 days after ASDH tended to be smaller in the Argatroban-treated group when compared to the vehicle group (8.1 ± 1.1 vs. 10.1 ± 2.3 mm2, n.s.). Motor deficits in the beam balance test were not significantly less severe in the Argatroban-treated group. Animals treated with SCH79797 also showed a trend towards dose-dependent decreased lesion volume in comparison to the vehicle-treated group (1 µg: 4.3 ± 0.7 mm3; 5 µg: 3.8 ± 1.1 mm3; vehicle: 6.5 ± 2.0 mm3, n.s). CONCLUSIONS: Thrombin inhibition in the subdural blood and local cerebral blockade of PAR-1 cause a tendency towards reduced lesion volume or functional recovery. All results show a trend in favor of the acute treatment on the outcome parameters. Our results suggests that thrombin could be an important blood-derived factor during acute subdural hemorrhage that translates its deleterious effects in concert with other blood-induced factors.

Evaluation of a New Multiparameter Brain Probe for Simultaneous Measurement of Brain Tissue Oxygenation, Cerebral Blood Flow, Intracranial Pressure, and Brain Temperature in a Porcine Model.

BACKGROUND: A novel multiparameter brain sensor (MPBS) allows the simultaneous measurement of brain tissue oxygenation (ptiO2), cerebral blood flow (CBF), intracranial pressure (ICP), and brain temperature with a single catheter. This laboratory investigation evaluates the MPBS in an animal model in relation to established reference probes. METHODS: The study group consisted of 17 juvenile male pigs. Four MPBS and four reference probes were implanted per pig and compared simultaneously. The measured parameters were challenged by standardized provocations such as hyperoxia, dobutamine, and norepinephrine application, hypercapnia and hypoxia in combination with and without a controlled cortical impact (CCI) injury. Mean values over 2 min were collected for predefined time points and were analyzed using Bland-Altman plots. RESULTS: The protocol was successfully conducted in 15 pigs of which seven received CCI. ICP and ptiO2 were significantly influenced by the provocations. Subtraction of MPBS from reference values revealed a mean difference (limits of agreement) of 3.7 (- 20.5 to 27.9) mm Hg, - 2.9 (- 7.9 to 2.1) mm Hg, and 5.1 (- 134.7 to 145.0) % for ptiO2, ICP, and relative CBF, respectively. CONCLUSIONS: The MPBS is a promising measurement tool for multiparameter neuromonitoring. The conducted study demonstrates the in vivo functionality of the probe. Comparison with standard probes revealed a deviation which is mostly analogous to other multiparameter devices. However, further evaluation of the device is necessary before it can reliably be used for clinical decision making.

The involvement of thrombin in the pathogenesis of glioblastoma.

Prothrombin and its active derivative thrombin are key members of the coagulation system. The only site of extra-hepatic thrombin expression is the central nervous system (CNS), where it is involved in brain development, protection, and regeneration. Thrombin affects various degenerative and ischemic CNS diseases like Alzheimer's, multiple sclerosis, cerebral ischemia, and hemorrhage in a dose dependent manner. Additionally, the association of thrombin with various malignancies has recently become evident. Thrombin facilitates the interaction between tumor cells with platelets, endothelial cells, and the adhesion to matrix proteins in various tumor types. Consequently, thrombin enables tumor cell seeding and metastasis, resulting in increased tumor cell growth and angiogenesis. Despite the exceptional position of thrombin in the CNS, its involvement in brain tumor course and development has so far been largely neglected. Over the last decade, several studies found a detrimental effect of thrombin in the most devastating of all primary brain tumors, glioblastomas (GBM). This review highlights the current knowledge on the involvement of thrombin in the pathophysiology and clinical course of GBMs. © 2017 Wiley Periodicals, Inc.

The Importance of Thrombin in Cerebral Injury and Disease.

There is increasing evidence that prothrombin and its active derivative thrombin are expressed locally in the central nervous system. So far, little is known about the physiological and pathophysiological functions exerted by thrombin in the human brain. Extra-hepatic prothrombin expression has been identified in neuronal cells and astrocytes via mRNA measurement. The actual amount of brain derived prothrombin is expected to be 1% or less compared to that in the liver. The role in brain injury depends upon its concentration, as higher amounts cause neuroinflammation and apoptosis, while lower concentrations might even be cytoprotective. Its involvement in numerous diseases like Alzheimer's, multiple sclerosis, cerebral ischemia and haemorrhage is becoming increasingly clear. This review focuses on elucidation of the cerebral thrombin expression, local generation and its role in injury and disease of the central nervous system.

Influence of Blood Components on Neuroinflammation, Blood-Brain Barrier Breakdown, and Functional Damage After Acute Subdural Hematoma in Rats.

A central component of injury development after acute subdural hematoma (ASDH) is the increased intracranial pressure and consecutive mechanical reduction of cerebral blood flow (CBF). However, the role of different blood constituents in ASDH as additional lesioning factors remains unclear. This study examines the influence of blood components on neuroinflammation, blood-brain barrier (BBB) breakdown, and functional deficits in a rat model of ASDH. We infused corpuscular (whole blood, whole blood lysate, and red cell blood) and plasmatic (blood plasma, anticoagulated blood plasma, and aqueous isotonic solution) blood components into the subdural space while CBF was monitored. Rats then underwent behavioral testing. Lesion analysis and immunohistochemistry were performed 2 days after ASDH. Inflammatory reaction was assessed using staining for ionized calcium-binding adaptor molecule 1 and glial fibrillary acidic protein, interleukin-1ß, tumor necrosis factor-alpha, and membrane attack complex. Integrity of the BBB was evaluated with albumin and matrix metalloproteinase 9 (MMP9) staining. We observed a significant drop in CBF in the corpuscular group (75% ± 7.5% of baseline) with distinct post-operative deficits and larger lesion volume compared to the plasmatic group (13.6 ± 5.4 vs. 1.3 ± 0.4 mm3). Further, inflammation was significantly increased in the corpuscular group with stronger immunoreaction. After whole blood infusion, albumin and MMP9 immunoreaction were significantly increased, pointing toward a disrupted BBB. The interaction between corpuscular and plasmatic blood components seems to be a key factor in the detrimental impact of ASDH. This interaction results in neuroinflammation and BBB leakage. These findings underscore the importance of performing surgery as early as possible and also provide indications for potential pharmacological targets.

Early Onset of Rapid Lesion Growth in an Acute Subdural Hematoma Model in Rats.

OBJECTIVE: Acute subdural hematoma (ASDH) leads to the highest mortality rates of all head injuries with secondary brain damage playing a pivotal role in terms of morbidity and mortality. In patients with ASDH, a delay in surgery leads to disproportional mortality. The benefit of (very) early therapy is therefore, a target of ongoing research. As the process of delayed brain damage in ASDH has not yet been described, this study therefore aimed to examine secondary lesion growth in an experimental rat model of ASDH to define the ideal timing for testing potential neuroprotective therapies. METHODS: Cerebral blood flow was monitored during ASDH induction with 300 µl of autologous blood. Lesion growth was characterized using Hematoxylin-Eosin- , Cresyl-Violet-, and Fluoro-Jade B-staining for early signs of neuronal degeneration. Histological evaluations were performed between 15 minutes and 24 hours after ASDH. RESULTS: There was a significant reduction of cerebral blood flow after ASDH. Fluoro-Jade B-positive cells were visible 15 minutes after ASDH in the lesioned hemisphere. Nonlinear growth of lesion volume from 3.7 ± 0.4 mm3 to 17.5 ± 0.6 mm3 was observed at 24 hours in Hematoxylin-Eosin-staining. CONCLUSIONS: The most damage develops between 15 minutes and 1 hour and again between 2 and 6 hours after ASDH. The time course of lesion growth supports the approach of early surgery for patients. It furthermore constitutes a basis for further ASDH research with more clearly defined time windows for therapy in animal models.

In-vivo time course of organ uptake and blood-brain-barrier permeation of poly(L-lactide) and poly(perfluorodecyl acrylate) nanoparticles with different surface properties in unharmed and brain-traumatized rats.

Background: Traumatic brain injury (TBI) has a dramatic impact on mortality and quality of life and the development of effective treatment strategies is of great socio-economic relevance. A growing interest exists in using polymeric nanoparticles (NPs) as carriers across the blood-brain barrier (BBB) for potentially effective drugs in TBI. However, the effect of NP material and type of surfactant on their distribution within organs, the amount of the administrated dose that reaches the brain parenchyma in areas with intact and opened BBB after trauma, and a possible elicited inflammatory response are still to be clarified. Methods: The organ distribution, BBB permeation and eventual inflammatory activation of polysorbate-80 (Tw80) and sodiumdodecylsulfate (SDS) stabilized poly(L-lactide) (PLLA) and poly(perfluorodecyl acrylate) (PFDL) nanoparticles were evaluated in rats after intravenous administration. The NP uptake into the brain was assessed under intact conditions and after controlled cortical impact (CCI). Results: A significantly higher NP uptake at 4 and 24 h after injection was observed in the liver and spleen, followed by the brain and kidney, with minimal concentrations in the lungs and heart for all NPs. A significant increase of NP uptake at 4 and 24 h after CCI was observed within the traumatized hemisphere, especially in the perilesional area, but NPs were still found in areas away from the injury site and the contralateral hemisphere. NPs were internalized in brain capillary endothelial cells, neurons, astrocytes, and microglia. Immunohistochemical staining against GFAP, Iba1, TNFα, and IL1ß demonstrated no glial activation or neuroinflammatory changes. Conclusions: Tw80 and SDS coated biodegradable PLLA and non-biodegradable PFDL NPs reach the brain parenchyma with and without compromised BBB by TBI, even though a high amount of NPs are retained in the liver and spleen. No inflammatory reaction is elicited by these NPs within 24 h after injection. Thus, these NPs could be considered as potentially effective carriers or markers of newly developed drugs with low or even no BBB permeation.

Microglia-Dependent and Independent Brain Cytoprotective Effects of Mycophenolate Mofetil During Neuronal Damage.

Acute lesions of the central nervous system often lead to permanent limiting deficits. In addition to the initial primary damage, accompanying neuroinflammation is responsible for progression of damage. Mycophenolate mofetil (MMF) as a selective inhibitor of inosine 5-monophosphate dehydrogenase (IMPDH) was shown to modulate the inflammatory response and promote neuronal survival when applied in specific time windows after neuronal injury. The application of brain cytoprotective therapeutics early after neuronal damage is a fundamental requirement for a successful immunomodulation approach. This study was designed to evaluate whether MMF can still mediate brain cytoprotection when applied in predefined short time intervals following CNS injury. Furthermore, the role of microglia and changes in IMPDH2 protein expression were assessed. Organotypic hippocampal slice cultures (OHSC) were used as an in vitro model and excitotoxically lesioned with N-methyl-aspartate (NMDA). Clodronate (Clo) was used to deplete microglia and analyze MMF mediated microglia independent effects. The temporal expression of IMPDH2 was studied in primary glial cell cultures treated with lipopolysaccharide (LPS). In excitotoxically lesioned OHSC a significant brain cytoprotective effect was observed between 8 and 36 h but not within 8 and 24 h after the NMDA damage. MMF mediated effects were mainly microglia dependent at 24, 36, 48 h after injury. However, further targets like astrocytes seem to be involved in protective effects 72 h post-injury. IMPDH2 expression was detected in primary microglia and astrocyte cell cultures. Our data indicate that MMF treatment in OHSC should still be started no later than 8-12 h after injury and should continue at least until 36 h post-injury. Microglia seem to be an essential mediator of the observed brain cytoprotective effects. However, a microglia-independent effect was also found, indicating involvement of astrocytes.

Multiparametric Monitoring of Early Pathophysiological Changes in a Porcine Model of Sequential Focal and Global Cerebral Ischemia.

OBJECTIVE: Large animal models of cerebral ischemia have the potential to increase the translational value of stroke research. This study aims to measure early changes of brain tissue oxygen pressure (ptiO2) and cerebral blood flow (CBF) to characterize a porcine model of sequential middle cerebral artery occlusion (MCAO) and common carotid artery occlusion (CCAO). METHODS: Eight juvenile German Landrace pigs received unilateral MCAO via a frontotemporal approach under continuous intraparenchymal multiparametric monitoring. Insufficient reduction (i.e., <50% in both ptiO2 and CBF) was followed by additional bilateral CCAO. Neurodegenerative changes were detected by Fluoro-Jade B (FJB) staining. RESULTS: Only 1 of 8 animals demonstrated a decrease of >50% in both ptiO2 and CBF after MCAO. Additional CCAO in 7 pigs led to a significant reduction of both ipsilateral and contralateral ptiO2 (P < 0.01) but not of CBF. There was no difference in ptiO2 and FJB positive area between hemispheres in this group. Measurement of ptiO2 correlated negatively with the FJB positive area (P < 0.05). CONCLUSIONS: Intraparenchymal multiparametric measurements of acute changes in ptiO2 and CBF were variable after MCAO. Bilateral CCAO led to a consistent decrease in ptiO2 and correlated with early degenerative histologic changes, but CBF did not. Real-time procedural ptiO2 monitoring could provide useful guidance in large animal ischemia models. Feasibility in the context of global cerebral hypoperfusion is demonstrated.

The Cerebral Thrombin System Is Activated after Intracerebral Hemorrhage and Contributes to Secondary Lesion Growth and Poor Neurological Outcome in C57Bl/6 Mice.

With increasing evidence for the existence of a cerebral thrombin system, coagulation factor IIa (thrombin) is suspected to influence the pathogenesis of secondary injury progression after intracerebral hemorrhage (ICH). We hypothesized that mechanisms associated with local volume expansion after ICH, rather than blood constituents, activate the cerebral thrombin system and are responsible for detrimental neurological outcome. To test this hypothesis, we examine the local thrombin expression after ICH in a C57BL/6N mouse model in the presence and absence of blood constituents. ICH was established using stereotaxic orthotopic injection of utologous blood (n = 10) or silicone oil as inert volume substance (n = 10) into the striatum. Intracranial pressure (ICP), cerebral blood flow (CBF), and mean arterial blood pressure (MAP) were monitored during and 30 min after the procedure. No significant differences between ICP, CBF, and MAP were found between both groups. Prothrombin messenger RNA expression was upregulated early after ICH. Immunohistochemistry showed an increase of perilesional thrombin in both groups (blood, 4.24-fold; silicone, 3.10-fold), whereas prothrombin fragment (F1.2) was elevated only in the absence of whole blood. Thrombin expression is colocalized with neuronal antigen expression. After 24 h, lesion size and neuronal loss were similar. Perihematomal thrombin correlated with increased neuronal loss and detrimental neurological outcome in vivo. In our study, we demonstrate, for the first time, that the local cerebral thrombin system is activated after ICH and that this activation is independent of the presence of whole-blood constituents. In our study, neuronal damage is driven by local thrombin expression and leads to an adverse clinical outcome.

High CSF thrombin concentration and activity is associated with an unfavorable outcome in patients with intracerebral hemorrhage.

BACKGROUND: The cerebral thrombin system is activated in the early stage after intracerebral hemorrhage (ICH). Expression of thrombin leads to concentration dependent secondary neuronal damage and detrimental neurological outcome. In this study we aimed to investigate the impact of thrombin concentration and activity in the cerebrospinal fluid (CSF) of patients with ICH on clinical outcome. METHODS: Patients presenting with space-occupying lobar supratentorial hemorrhage requiring extra-ventricular drainage (EVD) were included in our study. The CSF levels of thrombin, its precursor prothrombin and the Thrombin-Antithrombin complex (TAT) were measured using enzyme linked immune sorbent assays (ELISA). The oxidative stress marker Superoxide dismutase (SOD) was assessed in CSF. Initial clot size and intraventricular hemorrhage (IVH) volume was calculated based on by computerized tomography (CT) upon admission to our hospital. Demographic data, clinical status at admission and neurological outcome were assessed using the modified Rankin Scale (mRS) at 6-weeks and 6-month after ICH. RESULTS: Twenty-two consecutive patients (9 females, 11 males) with supratentorial hemorrhage were included in this study. CSF concentrations of prothrombin (p < 0.005), thrombin (p = 0.005) and TAT (p = 0.046) were statistical significantly different in patients with ICH compared to non-hemorrhagic CSF samples. CSF concentrations of thrombin 24h after ICH correlated with the mRS index after 6 weeks (r2 = 0.73; < 0.005) and 6 months (r2 = 0.63; < 0.005) after discharge from hospital. Thrombin activity, measured via TAT as surrogate parameter of coagulation, likewise correlated with the mRS at 6 weeks (r2 = 0.54; < 0.01) and 6 months (r2 = 0.66; < 0.04). High thrombin concentrations coincide with higher SOD levels 24h after ICH (p = 0.01). CONCLUSION: In this study we found that initial thrombin concentration and activity in CSF of ICH patients did not correlate with ICH and IVH volume but are associated with a poorer functional neurological outcome. These findings support mounting evidence of the role of thrombin as a contributor to secondary injury formation after ICH.

Occurrence of Spontaneous Cortical Spreading Depression Is Increased by Blood Constituents and Impairs Neurological Recovery after Subdural Hematoma in Rats.

Acute subdural hemorrhage (ASDH) is common and associated with severe morbidity and mortality. To date, the role of spontaneous cortical spreading depression (sCSD) in exaggerating secondary injury after ASDH, is poorly understood. The present study contains two experimental groups: First, we investigated and characterized the occurrence of sCSD after subdural blood infusion (300 µL) via tissue impedance (IMP) measurement in a rat model. Second, we compared the occurrence and influence of sCSD on lesion growth and neurological deficit in the presence and absence of whole blood constituents. In the first experimental group, three IMP traits could be distinguished after ASDH: no sCSD, recurrent sCSD, and constant elevated IMP (anoxic depolarization [AD]). In the second experimental group, sCSD occurred more often after autologous blood, compared with paraffin oil infusion. Lesion volume 7 days post-ASDH was 27.3 ± 6.8 mm3 after blood and 3.4 ± 2.1 mm3 after paraffin oil infusion. Subgroup analysis showed larger lesion size in animals with sCSD, than in those without. Further, occurrence of sCSD led to worse neurological outcomes in both groups. sCSD occurs early after ASDH and does not depend on the presence of whole blood constituents. However, numbers and degree of sCSD are more frequent and severe after autologous blood infusion, compared with an inert volume substance. The occurrence of sCSD leads to lesion growth and worse neurological outcome. Thus, our data advocate close monitoring and targeted treatment of sCSD after ASDH evacuation.

Emboli formation rather than inflammatory mediators are responsible for increased cerebral water content after conventional and assisted beating-heart myocardial revascularization in a porcine model.

BACKGROUND AND PURPOSE: Emboli and proinflammatory mediators are suspected of generating cerebral edema after coronary surgery. In contrast to cardiopulmonary bypass (CPB), off-pump coronary artery bypass surgery (OPCAB) reduces microemboli count and proinflammatory mediator release but carries the risk of hemodynamic instability. A microaxial blood pump can augment cardiac output. METHODS: Coronary bypasses were constructed in pigs with CPB and cardioplegia (n=9), OPCAB (n=9), or blood-pump support CAB (n=9). Nine animals underwent sham operation. Embolus count was monitored and regional cerebral blood flow was assessed with microspheres in 21 brain specimens per animal (n=189 per group). Interleukins 6 and 8 and tumor necrosis factor-alpha concentrations were determined. These variables were studied before, during, and for 4 hours after surgery. Finally, cerebral water content was determined. RESULTS: During CPB and blood-pump CAB, a significant number of emboli were counted in contrast to OPCAB and controls (P<0.05). During CPB, regional cerebral blood flow was affected (32 of 189) and showed reactive hyperemia except in 10 specimens after aortic cross-clamp release. This impairment persisted in 20 specimens. During and after OPCAB, regional cerebral blood flow remained nearly unchanged but showed low flow during (58 of 189) and after (35 of 189) the blood-pump run. A significant increase in proinflammatory mediators was observed only in the CPB group. CPB and blood-pump CAB significantly increased cerebral water content (P<0.05). A strong correlation between embolic load and cerebral water content was observed in all groups. No correlation between proinflammatory mediator release and cerebral water content was detected. CONCLUSIONS: Emboli formation rather than inflammatory mediators are responsible for increased cerebral water content after conventional and assisted beating-heart myocardial revascularization.

Effects of hypertonic/hyperoncotic treatment and surgical evacuation after acute subdural hematoma in rats.

OBJECTIVE: The treatment of acute subdural hematoma (ASDH) consists mainly of surgical evacuation of the hematoma. It is conceivable that early preoperative neuroprotection with hypertonic/hyperoncotic treatment (HHT) can improve survival rates. The present study investigated the benefit of treatment with hypertonic/hyperoncotic solution on functional and histologic outcome as supportive therapy accompanying surgical intervention. DESIGN: Laboratory experiment. SETTING: University laboratory. SUBJECTS: Male Sprague-Dawley rats weighing 296-350 g (n = 56). INTERVENTIONS: ASDH was induced through subdural infusion of 400 microL of autologous venous blood. Thirty minutes after subdural blood infusion, the rats received either HyperHAES (7.2% saline/6% hydroxyethyl starch) or vehicle (NaCl 0.9%) intravenously, followed by surgical evacuation of the hematoma 1 hr after ASDH induction in those rats scheduled for surgical treatment. The experiment was divided into two parts: an acute study, which explored acute effects of HHT on blood variables, ASDH-induced changes of intracranial pressure (ICP), and cerebral perfusion pressure (CPP), and a chronic study, which investigated the chronic effects of HHT, surgical blood clot evacuation, and the combination of both on the functional and histologic outcome following ASDH (12 days). MEASUREMENTS AND MAIN RESULTS: In the acute study, HHT expectedly raised the serum sodium concentration and lowered hematocrit. ASDH increased ICP and decreased CPP in all groups. HHT improved CPP by reducing ICP. In the chronic study, all treated groups showed a better recovery with respect to neurologic function and neuronal cell death compared with the vehicle-treated ASDH group. HHT with surgical evacuation or HHT alone improved functional and histologic outcome slightly more than surgical evacuation alone. CONCLUSIONS: In this rat model, HHT led to a decrease of ICP after ASDH. This significantly improved functional and histologic outcome, which was comparable to the effects after blood evacuation alone. The combination of evacuation of subdural blood and early HHT improved histologic outcome further but not significantly, which was due to the strong effects of single treatments and a ceiling effect of the combined treatment in this model.

Acute subdural hematoma in pigs: role of volume on multiparametric neuromonitoring and histology.

Traumatic brain injury (TBI) is often complicated by acute subdural hemorrhage (ASDH) with a high mortality rate. The pathophysiological mechanisms behind such an injury type and the contribution of blood to the extent of an injury remain poorly understood. Therefore, the goals of this study were to establish a porcine ASDH model in order to investigate pathomechanisms of ASDH and to compare effects induced by blood or sheer volume. Thus, we infused 2, 5, and 9 mL of blood (up to 15% of intracranial volume), and we compared a 5-mL blood and paraffin oil volume to separate out effects of extravasated blood on brain tissue. An extended neuromonitoring was applied that lasted up to 12 h after injury and included intracranial pressure (ICP), cerebral perfusion pressure (CPP), tissue oxygen concentration (ptiO(2)), biochemical markers (glutamate, lactate), somatosensory evoked potentials (SEP), brain water content, and histological assessment (Lesion Index [LI]). Volume-dependent changes were detected mainly during the first hours after injury. ICP increased to significant levels (p < 0.05) of 36.89 +/- 1.59, 15.52 +/- 0.48, and 11.25 +/- 0.35 mm Hg after 9, 5, and 2 mL of subdural blood, respectively (sham, 4.85 +/- 0.06 mm Hg). The ptiO(2) dropped drastically after 9 mL of subdural blood without recovery in both hemispheres to below 20% of baseline, but was affected little after 2 and 5 mL in the acute monitoring period (maximal drop to 71% of baseline). Later, 5 mL of blood led to a significant increase of ptiO(2) compared to 2 mL ipsilaterally (p < 0.05). Glutamate and lactate showed a comparable pattern with a long-lasting increase after 9 mL of blood and short-lasting changes after 2 and 5 mL. The two smaller volumes caused an increased brain swelling (2 mL, 80.60 +/- 0.34%; 5 mL, 81.20 +/- 0.66%; p < 0.05 vs. sham), a significant LI (sham, 6.4 +/- 1.4; 2 mL, 30.0 +/- 0.95; 5 mL, 32.1 +/- 1.2; p < 0.05 vs. sham), and a reduced SEP amplitude (5 mL, p < 0.05 vs. baseline) at the end of the experiment. A 9-mL led to herniation during the experiment causing dramatical brain swelling and acute histological damage. Comparison of blood volume with paraffin oil showed no significance, indicating that volume alone determines the acute pathophysiological processes leading to a rapidly developing histological damage. Additional effects due to blood contact with brain tissue (e.g., inflammation) may be detected only at later time points (>12 h).

Comparative Examination of Temporal Glyoxalase 1 Variations Following Perforant Pathway Transection, Excitotoxicity, and Controlled Cortical Impact Injury.

Following acute neuronal lesions, metabolic imbalance occurs, the rate of glycolysis increases, and methylglyoxal (MGO) forms, finally leading to metabolic dysfunction and inflammation. The glyoxalase system is the main detoxification system for MGO and is impaired following excitotoxicity and stroke. However, it is not known yet whether alterations of the glyoxalase system are also characteristic for other neuronal damage models. Neuronal damage was induced in organotypic hippocampal slice cultures by transection of perforant pathway (PPT; 5 min to 72 h) and N-methyl-D-aspartate (NMDA; 50 µM for 4 h) or in vivo after controlled cortical impact (CCI) injury (2 h to 14 days). Temporal and spatial changes of glyoxalase I (GLO1) were investigated by Western blot analyses and immunohistochemistry. In immunoblot, the GLO1 protein content was not significantly affected by PPT at all investigated time points. As described previously, NMDA treatment led to a GLO1 increase 24 and 48 h after the lesion, whereas PPT increased GLO1 immunoreactivity within neurons only at 48 h postinjury. Immunohistochemistry of brain tissue subjected to CCI unveiled positive GLO1 immunoreactivity in neurons and astrocytes at 1 and 3 days after injury. Two hours and 14 days after CCI, no GLO1 immunoreactivity was observed. GLO1 protein content changes are associated with excitotoxicity but seemingly not to fiber transection. Cell-specific changes in GLO1 immunoreactivity after different in vitro and in vivo lesion types might be a common phenomenon in the aftermath of neuronal lesions.

Cerebral embolic ischemia in rats: correlation of stroke severity and functional deficit as important outcome parameter.

The embolic MCA occlusion model in rats is used for recanalisation studies in acute stroke. In addition to the determination of lesion size, the assessment of functional outcome may improve the value of this model. Male Wistar rats were submitted to MCA clot embolism or sham surgery. In order to achieve a larger variety of lesion volume, 2 subgroups (each 7 animals) were subjected to differently sized emboli (30 and 40 mm). Follow-up period was 6 days. Outcome assessment consisted of a test battery including parallel bar crossing, observation of behaviour in an open field and an 8-arm maze and a neurological score with ten different sensorimotor and coordinative items. Animals were perfusion-fixed on day 7 (blinded examination). For both subgroups, there were significant impairments with regard to performance on the Neuro score, parallel bar crossing and maze exploration. Improvement was only partial during the follow-up period. On follow-up day 6, there was still a significant correlation between total infarct volume and functional outcome on the Neuro score (R=0.80, p=0.0006) and the exploration behaviour in the maze (R=0.66, p=0.01). Application of emboli with a length of 40 mm caused more functional impairment and a more extended lesion volume compared with 30 mm. We present outcome tests that provide quantitative and objective tools to test functional impairment in rats following embolic stroke.

Spreading depression and focal venous cerebral ischemia enhance cortical neurogenesis.

Endogenous neurogenesis can arise from a variety of physiological stimuli including exercise, learning, or "enriched environment" as well as pathological conditions such as ischemia, epilepsy or cortical spreading depression. Whether all these conditions use a common trigger to set off endogenous neurogenesis is yet unclear. We hypothesized that cortical spreading depression (CSD) induces neurogenesis in the cerebral cortex and dentate gyrus after cerebral venous ischemia. Forty-two Wistar rats alternatively underwent sham operation (Sham), induction of ten CSDs or venous ischemia provoked via occlusion of two adjacent superficial cortical vein followed by ten induced CSDs (CSD + 2-VO). As an additional control, 15 naïve rats received no intervention except 5-bromo-2'-deoxyuridine (BrdU) treatment for 7 days. Sagittal brain slices (40 µm thick) were co-stained for BrdU and doublecortin (DCX; new immature neuronal cells) on day 9 or NeuN (new mature neuronal cells) on day 28. On day 9 after sham operation, cell proliferation and neurogenesis occurred in the cortex in rats. The sole induction of CSD had no effect. But on days 9 and 28, more proliferating cells and newly formed neurons in the ipsilateral cortex were observed in rats subjected to CSD + 2VO than in rats subjected to sham operation. On days 9 and 28, cell proliferation and neurogenesis in the ipsilateral dentate gyrus was increased in sham-operated rats than in naïve rats. Our data supports the hypothesis that induced cortical neurogenesis after CSD + 2-VO is a direct effect of ischemia, rather than of CSD alone.

Blood Aggravates Histological and Functional Damage after Acute Subdural Hematoma in Rats.

Acute subdural hematoma (ASDH) is associated with high morbidity and mortality. Whether the volume effect of the hematoma and increase of intracranial pressure (ICP) or the local effect of blood are responsible for this severe pathophysiology is unclear. Therefore, we compared subdural infusion of autologous blood and paraffin oil in a rat model of ASDH. In a histological study, we investigated the effects on acute ICP, cerebral perfusion pressure (CPP), cerebral blood flow (CBF), tissue oxygen changes, and brain damage at 2, 24, and 96 h post-infusion. Inflammatory reaction was analyzed by immuno-staining for microglia (ionized calcium binding adaptor molecule 1 [Iba1]) and activated astrocytes (glial fibrillary acidic protein [GFAP]). Besides acute ICP and CBF changes, we investigated the development of behavior (neuroscore and beamwalk test) for up to 4 days after injury in a behavioral study. Despite comparably increased ICP, there was a more pronounced lesion growth in the blood infusion group during the first 96 h. Further, there was an increased peri-lesional immunoreactive area of Iba1 and GFAP 96 h post-infusion, primarily in the blood infusion group, whereas hippocampal damage was comparable in both infusion groups. In the behavioral evaluation, paraffin-infused animals showed a better recovery, compared with the blood infusion group. In conclusion, comparable acute time-course of ICP, CPP, and CBF clearly indicates that the differences in lesion size, inflammatory reaction, and behavioral deficits after blood- and paraffin oil-induced ASDH are partially due to blood constituents. Therefore, current data suggest that subdural hematomas should be completely removed as quickly as possible; decompression alone may not be sufficient to prevent secondary brain damage.