Robert E. Tibbs

Bilirubin produces apoptosis in cultured bovine brain endothelial cells

Blood components such as oxyhemoglobin are believed to cause cerebral vasospasm by inducing contraction and cell death in cerebral arteries. We have observed previously that oxyhemoglobin produces apoptotic changes in cultured endothelial cells. This study was undertaken to explore if bilirubin, a bi-product of hemoglobin degradation, will produce similar cytotoxicity in endothelial cells. Cultured bovine brain microvascular endothelial cells were incubated in four concentrations of bilirubin (10, 25, 50, and 100 microM) for varying times (6, 12, and 24 h). Control cells were incubated in saline or vehicle (NaOH solution, <0.01% of 0.01 N) for similar time periods. The cultured cells were then observed microscopically for evidence of cellular alterations. Bilirubin (10-100 microM) produced apoptosis that appeared time-dependent but not clearly concentration-dependent. Biochemical markers for apoptosis such as DNA fragmentation and PARP cleavage were induced by bilirubin. We conclude that endothelial cells may undergo apoptosis after exposure to bilirubin.

Optison (FS069) disrupts the blood-brain barrier in rats.

Optison is a new echocardiographic contrast agent, designed for IV injection, that is very useful in delineating cardiac structures during ultrasound examination. Because Optison could be a valuable adjunct in the diagnosis and evaluation of congenital heart disease, this study was undertaken to assess its effects on the blood-brain barrier when introduced directly in the cerebral circulation, as might occur with some congenital lesions. In this study, Sprague-Dawley rats were anesthetized, and Optison, at various dosages, was injected into the carotid artery. After this, Evans blue dye, a marker for blood-brain barrier disruption, was injected at different time intervals. Gross and histologic examination of the animals’ brains revealed disruption of the blood-brain barrier that appeared to be Optison-dosage-dependent. Although the mechanism for this disruption is unclear, it may be related to the use of octofluoropropane gas used in the Optison as a contrast medium. Further studies are necessary to determine the pathologic consequences of Optison’s effects on the blood-brain barrier. Implications Optison appears to disrupt the blood-brain barrier when introduced directly into the cerebral arterial circulation. This may be related to the octafluoropropane gas used in Optison as a contrast medium. Optison should be used with caution when the possibility of a right-to-left shunt exists.

Morphological changes of cerebral penetrating arteries in a canine double hemorrhage model.

BACKGROUND Morphological presentations of cerebral vasospasm, such as dystrophy and desquamation of endothelial cells, corrugation of the internal elastic layer, and necrotic changes in smooth muscle cells, are well defined in large cerebral arteries. This study was undertaken to examine pathological changes in cerebral penetrating arteries in a canine double hemorrhage model. METHODS Eighteen mongrel dogs were subjected to an autologous arterial blood (0.4 mL/kg) injection into the cisterna magna on day 0 and day 2 after withdrawal of an equivalent amount of cerebrospinal fluid. Angiogram was performed on day 0 before the blood injection and on the day the dogs were sacrificed. The dogs were divided into four groups: control (day 0) (n = 4), hemorrhage and sacrificed on day 3 (n = 4), day 5 (n = 5), and day 7 (n = 5). The penetrating arteries were removed and found to be spastic on days 3, 5, and 7, but not in the control group. RESULTS Endothelial dystrophy and partial desquamation were recorded in all dogs sacrificed on days 5 and 7. Condensation of chromatin, blebbing of the membrane, and condensation of cytoplasm were identified in many endothelial cells, features that are consistent with apoptosis. The morphological changes in the penetrating arteries were more pronounced on days 5 and 7. CONCLUSIONS Vasospasm occurred in cerebral penetrating arteries in a canine double hemorrhage model. The morphological change in penetrating arteries, especially apoptosis in endothelial cells, is consistent with an early phase of vasospasm. Vasospasm in a penetrating artery may contribute to the cerebral ischemia that occurs during vasospasm.

The child as a projectile

Unintentional injury is the leading cause of death in children under the age of fourteen. The majority of these injuries/deaths occur when the child becomes airborne during an accident. The most common mechanisms by which children become airborne are motor vehicle collisions, bicycling accidents, and falls. A head injury is seen in a significant number of children in this setting. This includes injury to the scalp, skull, coverings of the brain, or the brain itself. These injuries are the most common cause of death in children resulting from unintentional injury. Other typical injuries include external bruises and abrasions, extremity fractures, and bruising or bleeding of internal organs. We propose to name this constellation of injuries the projectile child syndrome. This refers to those injuries occurring in infants and children as a result of becoming airborne during the events of an accident. The pattern of injuries seen as related to the anatomy of the child is stressed. A review of the impact to society and guidelines for prevention are presented. Anat. Rec. (New Anat.) 253:167–175, 1998.

Prevention of vasospasm in penetrating arteries with MAPK inhibitors in dog double-hemorrhage model

BACKGROUND Vasospasm in the penetrating arteries contributes to ischemic neurological deficit. It may be as important as angiographic vasospasm because it would explain the discrepancies between angiographic vasospasm and clinical symptoms in some patients. It may also underlie the different effects of vasodilators. The present study examined this hypothesis by looking at the effect of the inhibitors of mitogen-activated protein kinase (MAPK) on vasospasm of the penetrating arteries. METHODS Twenty-two adult mongrel dogs of either sex were used for the dog double-hemorrhage model. The dogs were randomly divided into four groups: control-hemorrhage, vehicle-treated, PD98059-treated, and U0126-treated groups. The drug injections were started on Day 3 after the first subarachnoid hemorrhage (SAH). The clinical status of the dogs was studied, based on their activity, appetite, and focal neurological symptoms. On Day 7, all the dogs were sacrificed, and the penetrating arteries from the brain stem were prepared for transmission electron microscopy. RESULTS (1) Severe vasospasm developed in the basilar arteries in the SAH-without-treatment group (control), in the DMSO-treated group (DMSO), and in the U0126 treatment group with mean reduction of the basilar artery diameter of 46.57%, 49.3%, and 39.6%, respectively. In the PD98059-treatment group only a mild vasospasm was observed and the mean reduction of the basilar artery diameter was 18.9%. (2) All the dogs in the control SAH and vehicle-treated groups developed severe angiographic and clinical vasospasm. The penetrating arteries were contracted, and the endothelial and smooth muscle cells were dystrophic. (3) The dogs in the U0126-treated group developed severe angiographic, but not clinical, vasospasm. The penetrating arteries were not contracted, and the endothelial and smooth muscle cells were not dystrophic. (4) The dogs in the PD98059 group developed mild angiographic vasospasm. No dog developed clinical symptoms that could be attributed to vasospasm. In morphological studies, the penetrating arteries were slightly contracted, but the cells were not dystrophic. CONCLUSIONS Vasospasm of the penetrating arteries, but not angiographic vasospasm, is consistent with the clinical symptoms and signs of vasospasm. MAPK may be important in maintaining vasospasm of both major and penetrating cerebral arteries. The correlation of the improvement in the clinical score with the reduction of vasospasm in the penetrating arteries demonstrated an important role of penetrating arteries in the morbidity and mortality caused by SAH.

Role of MAPK in chronic cerebral vasospasm

This study was undertaken to investigate the role of p44/42 MAPK in a dog double hemorrhage model of subarachnoid hemorrhage (SAH), and whether MEK inhibitors can alter the degree of SAH-induced vasoconstriction. The diameter of the basilar artery, which was compared with day 0 angiogram, decreased gradually in a time-dependent manner from day 3 (80%), day 5 (68%) through day 7 (53.5%). The level of MAPK (p44/42) immunoprecipitation peaked on day 3 and remained enhanced through day 7 (P < 0.05). MEK inhibitor PD98059 significantly reduced p44/42 MAPK immunoprecipitation and significantly reversed vasospasm and increased residual diameter to 79.0% on day 7. These results demonstrated that p44/42 MAPK kinase is involved in the pathogenesis of cerebral vasospasm. The MEK inhibitor PD98059 might be useful in the treatment of vasospasm.

Metabolic alterations in cerebrospinal fluid from double hemorrhage model of dogs

Abstract Even though cerebral vasospasm after subarachnoid hemorrhage (SAH) causes cerebral ischemia or infarction, the metabolic alterations in cerebrospinal fluids (CSF) after SAH have not been studied. This study was undertaken to measure the levels of glucose, lactate, pyruvate and glutamate in CSF from double hemorrhage dog models. Thirty-two mongrel dogs of either sex, weighing 18-24 kg, underwent double hemorrhage by percutaneous needle puncture of the cisterna magna and injection of autologous blood on day 0 and day 2. The dogs were then sacrificed on day 3, 5 and 7, after collecting CSF. In another study, the dogs were treated with mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126, and caspase-2 and caspase-3 inhibitors from day 3 to day 6 after initial blood injection. CSF was collected on day 7 before dogs were sacrificed. The concentration of glucose, lactate, pyruvate and glutamate in CSF was measured by photometrical method. Compared with CSF collected on day 0, glucose was decreased on days 5-7, lactate was increased on days 2-7, pyruvate was increased on days 2-7, and glutamate was increased on days 3-7 (p < 0.05). In the groups treated with MAPK or caspase inhibitors, most of the metabolic alterations remained unchanged as compared with CSF from untreated dogs. Clinically, caspase inhibitors-2 and-3, and MAPK inhibitor U0126 all failed to prevent vasospasm. MAPK inhibitor PD98059 partially prevented vasospasm. Our data demonstrated a metabolic alteration of glucose, glutamate, lactate and pyruvate in CSF during cerebral vasospasm. This metabolic change is consistent with the time course of cerebral vasospasm. This study suggests that brain energy metabolites and excitative amino acids are altered during cerebral vasospasm. [Neurol Res 2001; 23: 87-92]

Apoptosis in Basilar Endothelial Cells in a Canine Double Hemorrhage Model

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is a combination of prolonged contraction and vessel remodeling that may lead to cerebral ischemia or infarction [2]. Morphological studies of cerebral arteries affected by vasospasm often show extensive necrosis of smooth-muscle cells [1] as well as desquamation and dystrophy of endothelial cells [7, 11]. The actual mechanisms of cellular death following cerebral vasospasm are not known. Recent studies of post-SAH vasospasm in this laboratory strongly suggest that endothelial cells may be dying through apoptotic pathways [8, 10].

Role of MAPK in chronic cerebral vasospasm.

Many extracellular stimuli (including growth factors, hormones, stress, elevated temperature and G-protein coupled receptor agonists) initialize the phosphorylation cascades leading to the activation of mitogen-activated protein kinase (p44/42 MAPK) [2]. p44/42 MAPK activation may lead to an expression of the early genes, such as c-jun or c-fos, thus leading to the cellular proliferation [3]. p44/42 MAPK can cause the phosphorylation of caldesmon, thus promoting prolonged contraction [1]. It has been reported in several in vitro studies that p44/42 MAPK is involved in the prolonged contraction of the cerebral arteries, caused by hemolysate [8] and endothelin-1 [9], the most important causative agents for vasospasm. The levels of c-fos, and c-jun were increased in the presence of hemolysate, reaching maximum expression between 30 and 60 minutes in rat aorta smooth muscle cells [7].

Apoptosis in Cerebral Vasospasm After Subarachnoid Hemorrhage

We hypothesize that the mechanism of cerebral vasospasm after subarachnoid hemorrhage may not be a delayed and prolonged vascular response to certain spasmogens but a more complicated cascade, including contraction, cell death, and proliferation. This study was undertaken to demonstrate the apoptotic cell death induced by oxyhemoglobin in cultured endothelial cells in vitro, apoptosis in endothelium in vivo in a canine double hemorrhage model of experimental subarachnoid hemorrhage, and apoptosis in cerebral endothelial cells in a patient who died of cerebral vasospasm. (1) Oxyhemoglobin-produced endothelial apoptotic cell was demonstrated by apoptotic bodies [transmission electron microscopy (TEM)] and DNA ladders. (2) Endothelial cell damage and detachment were observed in dog middle cerebral arteries 5 days after blood injection. (3) In a patient who died 20 days after aneurysmal subarachnoid hemorrahge, endothelial detachment and apoptotic endothelial cells were demonstrated by TEM and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) reaction. These studies demonstrated that apoptosis may be an important step in vasospasm development. They opened a new avenue for treatment of vasospasm.