Controlled transient hypercapnia: a novel approach for the treatment of delayed cerebral ischemia after subarachnoid hemorrhage?

OBJECT: The authors undertook this study to investigate whether the physiological mechanism of cerebral blood flow (CBF) regulation by alteration of the arterial partial pressure of carbon dioxide (PaCO2) can be used to increase CBF after aneurysmal subarachnoid hemorrhage (aSAH). METHODS: In 6 mechanically ventilated patients with poor-grade aSAH, the PaCO2 was first decreased to 30 mm Hg by modification of the respiratory rate, then gradually increased to 40, 50 and 60 mm Hg for 15 minutes each setting. Thereafter, the respirator settings were returned to baseline parameters. Intracerebral CBF measurement and brain tissue oxygen saturation (StiO2), measured by near-infrared spectroscopy (NIRS), were the primary and secondary end points. Intracranial pressure (ICP) was controlled by external ventricular drainage. RESULTS: A total of 60 interventions were performed in 6 patients. CBF decreased to 77% of baseline at a PaCO2 of 30 mm Hg and increased to 98%, 124%, and 143% at PaCO2 values of 40, 50, and 60 mm Hg, respectively. Simultaneously, StiO2 decreased to 94%, then increased to 99%, 105%, and 111% of baseline. A slightly elevated delivery rate of cerebrospinal fluid was noticed under continuous drainage. ICP remained constant. After returning to baseline respirator settings, both CBF and StiO2 remained elevated and only gradually returned to pre-hypercapnia values without a rebound effect. None of the patients developed secondary cerebral infarction. CONCLUSIONS: Gradual hypercapnia was well tolerated by poor-grade SAH patients. Both CBF and StiO2 reacted with a sustained elevation upon hypercapnia; this elevation outlasted the period of hypercapnia and only slowly returned to normal without a rebound effect. Elevations of ICP were well compensated by continuous CSF drainage. Hypercapnia may yield a therapeutic potential in this state of critical brain perfusion. Clinical trial registration no.: NCT01799525 ( ClinicalTrials.gov ).

Brain edema formation and complement activation in a rat model of subarachnoid hemorrhage.

Previous studies have demonstrated that erythrocyte lysis and brain iron overload contribute to early brain injury after subarachnoid hemorrhage (SAH). Activation of the complement system and formation of the membrane attack complex can result in erythrocyte lysis and might, therefore, participate in such injury. This study, therefore, examined complement activation, blood-brain barrier (BBB) disruption, and brain edema in a rat SAH model.Subarachnoid hemorrhage was induced using a modified endovascular perforation technique. Brain complement activation was determined by Western blotting and immunohistochemistry. Brain edema was measured by dry/wet weight and BBB permeability assessed by measuring brain albumin levels.We found that there was expression of the membrane attack complex and clusterin in the frontal basal cortex and clot after SAH. The protein levels of the membrane attack complex were much higher in the frontal basal cortex at 72 h after SAH than those in sham (p < 0.01). We also found that brain water content was increased (81.9 ± 1.4 vs. 79.1 ± 0.2 % in sham, p < 0.05) and BBB was disrupted (albumin content: 10,695 ± 865 vs. 4,935 ± 3,121 pixels in sham, p < 0.01) 24 h after SAH.Our results suggest that complement activation after SAH might contribute to brain edema formation and BBB disruption after SAH.

A modified double injection model of cisterna magna for the study of delayed cerebral vasospasm following subarachnoid hemorrhage in rats.

Delayed cerebral vasospasm following subarachnoid hemorrhage (SAH) is a serious medical complication, characterized by constriction of cerebral arteries leading to varying degrees of cerebral ischemia. Numerous clinical and experimental studies have been performed in the last decades; however, the pathophysiologic mechanism of cerebral vasospasm after SAH still remains unclear. Among a variety of experimental SAH models, the double hemorrhage rat model involving direct injection of autologous arterial blood into the cisterna magna has been used most frequently for the study of delayed cerebral vasospasm following SAH in last years. Despite the simplicity of the technique, the second blood injection into the cisterna magna may result in brainstem injury leading to high mortality. Therefore, a modified double hemorrhage model of cisterna magna has been developed in rat recently. We describe here step by step the surgical technique to induce double SAH and compare the degree of vasospasm with other cisterna magna rat models using histological assessment of the diameter and cross-sectional area of the basilar artery.

Value of Perfusion CT, Transcranial Doppler Sonography, and Neurological Examination to Detect Delayed Vasospasm after Aneurysmal Subarachnoid Hemorrhage.

Background. If detected in time, delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) may be treated by balloon angioplasty or chemical vasospasmolysis in order to enhance cerebral blood flow (CBF) and protect the brain from ischemic damage. This study was conceived to compare the diagnostic accuracy of detailed neurological examination, Transcranial Doppler Sonography (TCD), and Perfusion-CT (PCT) to detect angiographic vasospasm. Methods. The sensitivity, specificity, positive and negative predictive values of delayed ischemic neurological deterioration (DIND), pathological findings on PCT-maps, and accelerations of the mean flow velocity (MVF) were calculated. Results. The accuracy of DIND to predict angiographic vasospasm was 0.88. An acceleration of MFV in TCD (>140 cm/s) had an accuracy of 0.64, positive PCT-findings of 0.69 with a higher sensitivity, and negative predictive value than TCD. Interpretation. Neurological assessment at close intervals is the most sensitive and specific parameter for cerebral vasospasm. PCT has a higher accuracy, sensitivity and negative predictive value than TCD. If detailed neurological evaluation is possible, it should be the leading parameter in the management and treatment decisions. If patients are not amenable to detailed neurological examination, PCT at regular intervals is a helpful tool to diagnose secondary vasospasm after aneurysmal SAH.

Deferoxamine reduces early brain injury following subarachnoid hemorrhage.

The effect of subarachnoid hemoglobin on neuroglial cells contributing to early brain injury is unclear. Several intracerebral hemorrhage studies indicated that pathological iron deposition in the brain contributes to secondary brain injury. Therefore, the purpose of this study was to investigate the relationship between iron and neuroglial cell changes following SAH, and examine the effect of deferoxamine (DFX). SAH was induced in male Sprague-Dawley rats (n = 56) using an endovascular perforation technique. Animals were treated with DFX (100 mg/kg) or vehicle for 3 days. Rats were sacrificed at 6 h, days 1 and 3 to determine non-heme iron and heme oxygenase (HO)-1 expression using Western blot and immunohistochemistry analysis. To assess neuronal cell death, Fluoro-Jade- and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stainings were performed. Marked HO-1 upregulation at day 3 (P < 0.01) was accompanied by elevated non-heme iron (P < 0.01) and ferritin levels (P < 0.01). DFX treatment reduced brain non-heme iron concentration, ferritin expression and neuronal cell death at day 3 (P < 0.01) following SAH. These results suggest that excessive hemoglobin and iron overload play an important role in early brain injury following SAH. Acute treatment with DFX significantly ameliorates neuronal cell death and may be a potential therapeutic agent for SAH.

Hypertrophy of the lumbar ligamentum flavum is associated with inflammation-related TGF-ß expression.

BACKGROUND: Despite the significance of hypertrophy of the ligamentum flavum (HLF) in the disease progress of neurogenic claudication, the cellular mechanisms underlying the gradual fibrotic thickening of the ligamentum flavum remain poorly understood. The aim of our study was to get insight into the contribution of inflammatory mechanisms to the development of hypertrophy. METHODS: Specimens of hypertrophied ligamenta flava were obtained at surgery from 20 patients with acquired lumbar osteoligamentous spinal canal stenosis from the central part of the ligament. Paraffin sections were stained with hematoxylin and eosin and Elastica van Gieson to evaluate extracellular matrix architecture, and immunohistochemistry was performed to characterize the inflammatory reaction and the sources of transforming growth factor beta (TGF-ß) expression. Sections of normal ligamenta flava obtained from corresponding anatomical sites and stained in parallel served as a control. RESULTS: HLF was characterized by a considerable distortion of the elastic matrix and fibrotic transformation by extracellular collagen deposition. All specimens showed highly inflammatory cellular infiltrates confined to regions exhibiting marked degeneration of the elastic matrix composed mainly of macrophages, scattered T lymphocytes, and neovascularization, thus representing a chronic inflammation. Surprisingly, macrophages as well as vascular endothelial cells but not fibroblasts showed a strong expression of TGF-ß, a strong inducer of extracellular collagen deposition. CONCLUSIONS: Macrophages were identified as a major cellular source of TGF-ß in advanced HLF and may perpetuate further hypertrophy. This finding suggests that modulating the immune response locally or systemically could prove to be effective for impeding the disease progress.

Hemoglobin and iron handling in brain after subarachnoid hemorrhage and the effect of deferoxamine on early brain injury.

The purpose of this study was to investigate hemoglobin and iron handling after subarachnoid hemorrhage (SAH), examine the relationship between iron and neuroglial cell changes, and determine whether deferoxamine (DFX) can reduce SAH-induced injury. The SAH was induced in Sprague-Dawley rats (n=110) using an endovascular perforation technique. Animals were treated with DFX (100 mg/kg) or vehicle 2 and 6 hours after SAH induction followed by every 12 hours for 3 days. Rats were killed at 6 hours, Days 1 and 3 to determine nonheme iron and examine iron-handling proteins using Western blot and immunohistochemistry. 8-Hydroxyl-2'-deoxyguanosine and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining were performed to assess oxidative DNA damage and neuronal cell death. After SAH, marked heme-oxygenase-1 (HO-1) upregulation at Day 3 (P<0.01) was accompanied by elevated nonheme iron (P<0.01), transferrin (Tf) (P<0.01), Tf receptor (P<0.05), and ferritin levels (P<0.01). Deferoxamine treatment reduced SAH-induced mortality (12% versus 29%, P<0.05), brain nonheme iron concentration, iron-handling protein expression, oxidative stress, and neuronal cell death at Day 3 (P<0.01) after SAH. These results suggest that iron overload in the acute phase of SAH causes oxidative injury leading to neuronal cell death. Deferoxamine effectively reduced oxidative stress and neuronal cell death, and may be a potential therapeutic agent for SAH.

Brain alpha- and beta-globin expression after intracerebral hemorrhage.

Our recent study has demonstrated that hemoglobin (Hb) is present in cerebral neurons and neuronal Hb is inducible after cerebral ischemia. In the present study, we examined the effects of intracerebral hemorrhage (ICH) on the mRNA levels of the α-globin (HbA) and the ß-globin (HbB) components of Hb and Hb protein in the brain in vivo and in vitro. In vivo, male Sprague-Dawley rats received either a needle insertion (sham) or an infusion of autologous whole blood into the basal ganglia and were killed at different time points. In vitro, cultured rat brain cells were used for HbA, HbB and Hb determination. Cultured neurons were exposed to 50 or 100 µM hemin for 24 h. Some neurons also were treated with deferoxamine, an iron chelator, or vehicle. Levels of HbA and HbB, Hb and hemopexin, a transporter of heme, were measured. We found that HbA, HbB and Hb are primarily expressed in neurons, with much lower expression in astrocytes and microglia. HbA, HbB and Hb expression in the perihematomal zone was increased after ICH and Hb was localized in neurons and glia. Hemin increased HbA, HbB and hemopexin mRNA levels in cultured neurons. Deferoxamine reduced hemin-induced neuronal Hb expression. ICH increased HbA and HbB expression in the brain, which may potentially serve to buffer the heme released during clot resolution.

Comparison of experimental rat models of early brain injury after subarachnoid hemorrhage.

OBJECTIVE: To investigate acute pathophysiological changes after subarachnoid hemorrhage (SAH) in rats and compare endovascular perforation and double blood injection models for studies of early brain injury after SAH. METHODS: Rat SAH was induced by endovascular perforation of the internal carotid artery (n = 41) or double injection of autologous blood into the cisterna magna (n = 23). Effects of SAH on arterial blood pressure, intracranial pressure, cerebral artery dimensions, and cerebral blood flow were measured. Neuronal death was assessed 24 hours after SAH. RESULTS: SAH was more severe in the endovascular perforation model (4-fold greater hemoglobin content on the basal brain surface), and mortality was greater (47%) than in the blood injection model (0%). Intracranial pressure increases were faster and greater in the perforation model. Correspondingly, cerebral blood flow reductions were greater after perforation than in the blood injection model, particularly in middle cerebral artery-supplied regions (32 +/- 16 versus 65 +/- 18 mL/100 g/min, P < 0.01). Diffuse neuronal death occurred in all rats in the perforation model but more seldom after blood injection. Anterior cerebral artery diameter and cross sectional area were significantly decreased on day 1 after SAH induction (52 +/- 21% and 22 +/- 16% of control values; P < 0.001) in the perforation model but not after blood injection. CONCLUSION: The perforation model produced more severe pathophysiological changes than the double blood injection, and it mimics human SAH in having an injured blood vessel and direct hemorrhagic brain lesion under arterial blood pressure. Therefore, endovascular perforation seems more suitable for study of acute SAH sequelae. However, further model refinement is required to reduce the high mortality rate.

Activated autophagy pathway in experimental subarachnoid hemorrhage.

Recent results have suggested a role for autophagy in acute brain injury but an involvement in subarachnoid hemorrhage (SAH) has not been investigated. Although, autophagy is a regulated process essential for cellular homeostasis, it may represent an additional type of cell death mechanism. This study employed a modified endovascular perforation rat model under guidance by intracranial pressure monitoring to investigate whether autophagy pathway is involved in the early brain injury following SAH. Sham-operated control rats underwent an identical procedure without vessel perforation. Electron microscopy was performed to examine the ultrastructural changes in neural cells after SAH. Additionally, microtubule-associated protein light chain-3 (LC3), cathepsin-D and beclin-1 were investigated by Western blot analysis and immunohistochemistry. Electron microscopically, there was a marked increase in autophagosomes and autolysosomes in neurons at Day 1 following SAH. Although LC3 could be detected in sham-operated control rats, the conversion of LC3-I to LC3-II was significantly increased at Day 1 (P<0.01) and Day 3 (P<0.05). The time-course of beclin-1 expression paralleled the LC3 conversion. Cathepsin-D expression was also elevated at Day 1 (P<0.01). Immunohistochemical study with antibodies against cathepsin-D and beclin-1 showed numerous positive stained cells after SAH, especially in deep layers of the fronto-basal cortex. Double immunolabeling revealed beclin-1 expression predominantly in neurons. This present study showed that the autophagy pathway is activated in neurons in the acute phase after SAH.

Effect of electrical stimulation of the cervical spinal cord on blood flow following subarachnoid hemorrhage.

OBJECT: Cervical spinal cord stimulation (SCS) increases global cerebral blood flow (CBF) and ameliorates cerebral ischemia according to a number of experimental models as well as some anecdotal reports in humans. Nonetheless, such stimulation has not been systematically applied for use in cerebral vasospasm. In the present study the authors examined the effect of cervical SCS on cerebral vasoconstriction in a double-hemorrhage model in rats. METHODS: Subarachnoid hemorrhage (SAH) was induced with 2 blood injections through an indwelling catheter in the cisterna magna. Spinal cord stimulation was applied 90 minutes after induction of the second SAH (Day 0) or on Day 5 post-SAH. Measurements of the basilar artery (BA) diameter and cross-sectional area and regional CBF (using laser Doppler flowmetry and (14)C-radiolabeled N-isopropyl-p-iodoamphetamine hydrochloride) were obtained and compared between SAH and sham-operated control rats that did not receive SCS. RESULTS: At Day 0 after SAH, there were slight nonsignificant decreases in BA diameter and cross-sectional area (89 +/- 3% and 81 +/- 4%, respectively, of that in controls) in no-SCS rats. At this time point, BA diameter and crosssectional area were slightly increased (116 +/- 6% and 132 +/- 9%, respectively, compared with controls, p < 0.001) in SCS-treated rats. On Day 5 after SAH, no-SCS rats had marked decreases in BA diameter and cross-sectional area (64 +/- 3% and 39 +/- 4%, respectively, compared with controls, p < 0.001) and corrugation of the vessel wall. These changes were reversed in rats that had received SCS (diameter, 110 +/- 9% of controls; area, 106 +/- 4% of controls; p < 0.001). Subarachnoid hemorrhage reduced CBF at Days 0 and 5 post-SAH, and SCS increased flows at both time points, particularly in regions supplied by the middle cerebral artery. CONCLUSIONS: Data in this study showed that SCS can reverse BA constriction and improve global CBF in this SAH model. Spinal cord stimulation may represent a useful adjunct in the treatment of vasospasm.

Characterization of an improved double hemorrhage rat model for the study of delayed cerebral vasospasm.

Rats have been shown in several studies to develop delayed cerebral vasospasm after subarachnoid hemorrhage (SAH). However, the rat model has not yet been fully validated, and there are number of methodological issues. In this study, we present an improved double hemorrhage rat model for studies of delayed cerebral vasospasm. Double hemorrhage was induced at an interval of 24h using a PE-10 catheter introduced into the cisterna magna through a parieto-occipital burr hole. Blood volumes consisted of 0.3/0.2 ml (Group 1) or 0.2/0.1 ml (Group 2). Regional cerebral blood flow (CBF) was measured with (14)C-IMP, and compared to a sham operated control group. Additionally, the diameter and cross-sectional area of basilar artery (BA) was measured microscopically at four different levels. Mortality in Group 1 was 40%, compared to 1.5% in Group 2 and further experiments focused on the latter. Immediately following the second hemorrhage, the vessel diameter and cross-sectional area of BA were not significantly changed, but by day 5 these had decreased to 64+/-3% and 39+/-4% of control (p<0.001), respectively. CBF changes were global, affecting all cerebral areas to a similar degree. Immediately following second SAH induction, CBF diminished to 62-73% of control (p<0.05). By day 1, CBF had slightly increased above normal (101-120%), but there was then a second progressive reduction in flow in all areas examined at day 5 reaching 70-85% of baseline (p<0.05). CBF then normalized by day 7 and 9. In this study, we present a new technique for creation of double SAH leading to delayed cerebral vasospasm in rats.

The effect of mitomycin C in reducing epidural fibrosis after lumbar laminectomy in rats.

OBJECT: Extensive epidural fibrosis after lumbar spine surgery might be an important underlying cause of failed-back syndrome. Based on previously obtained data, the effect of mitomycin C (MMC) in a concentration of 0.1 mg/ml on spinal epidural fibrosis in a rat laminectomy model was investigated in a large series. METHODS: Eighty adult Wistar rats underwent lumbar laminectomy. In 40 rats, MMC in a concentration of 0.1 mg/ml was locally applied to the laminectomy sites. No similar treatment was performed in the other 40 rats. At intervals from one to 12 weeks after laminectomy, both macroscopic and histological evaluations were performed. For radiological investigation, 10 rats underwent magnetic resonance (MR) imaging at 6 weeks postoperatively. Furthermore, the concentration of MMC in cerebrospinal fluid (CSF) and serum was determined 12 hours postoperatively in seven rats. Due to ease of absorption, high levels of MMC were rapidly detectable in serum, whereas the values obtained from the CSF were markedly lower. In the majority of MMC-treated laminectomy sites, epidural scarring was significantly reduced and dural adhesions were absent, in comparison with control sites (p < 0.001), as confirmed by MR images. Accordingly, the macroscopic dissection of epidural fibrous tissue to reexpose the dura mater was performed more easily and without severe bleeding in these rats. The healing of skin and the lumbar fascia was not affected, and dural leakage was not observed. All control sites showed dense epidural fibrosis with marked dural adherence. CONCLUSIONS: In this experimental model, it was shown that locally applied MMC in a concentration of 0.1 mg/ml effectively reduces epidural fibrosis and dural adherence without side effects in rats that underwent lumbar laminectomy.

The role of mitomycin C in reducing recurrence of epidural fibrosis after repeated operation in a laminectomy model in rats.

OBJECT: Extensive peridural fibrosis after lumbar spine surgery may contribute to poor outcome and recurrent symptoms leading to repeated operation. Secondary procedures are considerably hampered by the presence of scar tissue. Moreover, after excision of the peridural scar, the fibrous tissue may recur, leading to unsuccessful surgical outcome. Mitomycin C (MMC), an alkylating antibiotic substance isolated from Streptomyces caespitosus, potentially suppresses fibroblast proliferation after surgical intervention. The authors investigated the effect of MMC on the reformation of epidural fibrosis in a laminectomy model in rats. METHODS: Twenty-four Wistar rats underwent a repeated lumbar laminectomy 3 months after the first operation. In 12 rats, MMC in a concentration of 1 mg/ml was locally applied to the laminectomy site. No treatment was performed in the control group of the other 12 rats. All rats underwent clinical evaluation. Mobility ratings and any evidence of neurological deficit were recorded. Twelve weeks after the second operation, the animals were killed for histological examination. The extent of epidural fibrosis and dural adherence was evaluated. All MMC-treated animals showed reduced epidural scarring, compared with the control group. In nine MMC-treated rats (75%), dural adhesions were moderate. In contrast, all control sites showed dense epidural fibrosis with marked dural adherence. No side effects of the treatment were observed. CONCLUSIONS: In this experimental study, MMC in a concentration of 1 mg/ml locally applied significantly reduced recurrence of epidural fibrosis and dural adhesions without any side effects after repeated spinal surgery in a laminectomy model in rats.

Various surgical treatments of chronic subdural hematoma and outcome in 172 patients: is membranectomy necessary?

BACKGROUND: The initial surgical management of chronic subdural hematoma (CSDH) is still controversial, and a standard therapy does not exist. Because of the advanced age and multiple medical problems of the patients, surgical therapy is frequently associated with complications. METHODS: A retrospective study was performed on 172 patients with CSDH, comparing the efficacy of three different primary surgical methods: drainage of hematoma through two different burr-holes without membranectomy (Group A, n = 38); enlarged craniectomy with a size of about 30 mm craniotomy with partial membranectomy and drainage (Group B, n = 121); and extended craniotomy with partial membranectomy and drainage (Group C, n = 13). RESULTS: Independent of surgical method, the general outcome of the patients was good. The rate of reoperation in the group of burr-hole drainage was 16%, slightly lower than in partial membranectomy with enlarged craniectomy or extended craniotomy with 18% and 23%, respectively. In patients with coagulopathy, the rate of reoperation was 41% (16/43), significantly higher than the rate in noncoagulopathic patients 12% (15/129). CONCLUSIONS: In this study, an extended surgical approach with partial membranectomy has no advantages regarding the rate of reoperation and the outcome. As initial treatment, burr-hole drainage with irrigation of the hematoma cavity and closed-system drainage is recommended. Extended craniotomy with membranectomy is now reserved for instances of acute rebleeding with solid hematoma.

Mitomycin C in preventing spinal epidural fibrosis in a laminectomy model in rats.

OBJECT: Extensive epidural fibrosis after lumbar surgery may be the underlying cause in most cases of failed-back surgery syndrome. Various materials have been used to prevent epidural fibrosis, but only moderate success has been shown. Mitomycin C, an alkylosing antibiotic substance isolated from Streptomyces caespitosus, potentially supresses fibroblast proliferation after surgery. In this study, the authors investigated the effect of mitomycin C by local application on spinal epidural fibrosis in a rat laminectomy model. METHODS: Five Wistar rats underwent laminectomy at cervical, thoracic, and lumbar levels. Based on data obtained from ophthalmological studies, mitomycin C was applied to the laminectomy sites in various concentrations (0.01, 0.05, and 0.1 mg/ml). One laminectomy site in each rat was left untreated and thus served as a control. Evoked potentials were measured pre- and postoperatively, and all rats underwent clinical evaluation. Mobility status and evidence of neurological deficit were recorded. Twelve weeks later, the rats were killed, and the spinal column, including surrounding muscle tissue, was removed en bloc, decalcified, and fixed in formaldehyde. Epidural fibrosis was evaluated histologically. In all mitomycin C-treated laminectomy sites, epidural scarring was significantly reduced compared with control sites. Remarkably, dural adhesions were absent in laminectomy defects treated with mitomycin C concentrations of 0.05 and 0.1 mg/ml. Moderate to marked epidural fibrosis with adhesion to the dura mater was noted at sites receiving 0.01 mg/ml of mitomycin C. All control sites showed dense epidural fibrosis with marked dura adherence. CONCLUSIONS: In this experimental model, mitomycin C applied locally at a concentration of 0.1 mg/ml effectively reduced epidural fibrosis, completely avoided dural adherence, and induced no side effects.