Genetic Determinants of Cerebral Arterial Adaptation to Flow-loading.

BACKGROUND: In animal models, flow-loading is a necessary and sufficient hemodynamic factor to express the Cerebral Aneurysm (CA) phenotype. Using a rat model, this study characterizes the molecular events that comprise the cerebral arterial response to flow-loading and reveals their significance relating to the CA phenotype. OBJECTIVE: To characterize the molecular events that underlie expansive remodeling of cerebral arteries in two genetically distinct inbred rat strains with differential susceptibility to flow-dependent cerebrovascular pathology. METHODS: Thirty-two rats underwent bilateral common carotid artery ligation (BCL) (n=16) or Sham Surgery (SS) (n=16). Nineteen days later, vertebrobasilar arteries were harvested, histologically examined and analyzed for mRNA and protein expression. Flow-induced changes in histology, mRNA and protein expression were compared between BCL and SS rats. Differences between aneurysm-prone (Long Evans, LE) and resistant (Brown Norway, BN) strains were evaluated. RESULTS: Basilar Artery (BA) medial thickness/luminal diameter ratio was significantly reduced in BCL rats, without significant differences between LE (2.02 fold) and BN (1.94 fold) rats. BCL significantly altered BA expression of mRNA and protein but did not affect blood pressure. Eight genes showed similarly large flow-induced expression changes in LE and BN rats. Twenty-six flow responsive genes showed differences in flow-induced expression between LE and BN rats. The Cthrc1, Gsta3, Tgfb3, Ldha, Myo1d, Ermn, PTHrp, Rgs16 and TRCCP genes showed the strongest flow responsive expression, with the largest difference between LE and BN rats. CONCLUSIONS: Our study reveals specific molecular biological responses involved in flow-induced expansive remodeling of cerebral arteries that may influence differential expression of flowdependent cerebrovascular pathology.

Creatine transporter deficiency leads to increased whole body and cellular metabolism.

Creatine (Cr) is a guanidino compound required for rapid replenishment of ATP in cells with a high-energy demand. In humans, mutations in the Cr transporter (CRT;SLC6A8) prevent Cr entry into tissue and result in a significant intellectual impairment, epilepsy, and aphasia. The lack of Cr on both the whole body and cellular metabolism was evaluated in Crt knockout (Crt (-/y) ) mice, a high-fidelity model of human CRT deficiency. Crt (-/y) mice have reduced body mass and, however, show a twofold increase in body fat. There was increased energy expenditure in a home cage environment and during treadmill running in Crt (-/y) mice. Consistent with the increases in the whole-body metabolic function, Crt (-/y) mice show increased cellular metabolism as well. Mitochondrial respiration increased in skeletal muscle fibers and hippocampal lysates from Crt (-/y) mice. In addition, Crt (-/y) mice had increased citrate synthase activity, suggesting a higher number of mitochondria instead of an increase in mitochondrial activity. To determine if the increase in respiration was due to increased mitochondrial numbers, we measured oxygen consumption in an equal number of mitochondria from Crt (+/y) and Crt (-/y) mice. There were no changes in mitochondrial respiration when normalized to mitochondrial number, suggesting that the increase in respiration observed could be to higher mitochondrial content in Crt (-/y) mice.

Endovascular management of posthemorrhagic cerebral vasospasm: indications, technical nuances, and results.

Posthemorrhagic cerebral vasospasm (PHCV) is a common problem and a significant cause of mortality and permanent disability following aneurysmal subarachnoid hemorrhage. While medical therapy remains the mainstay of prevention against PHCV and the first-line treatment for symptomatic patients, endovascular options should not be delayed in medically refractory cases. Although both transluminal balloon angioplasty (TBA) and intra-arterial vasodilator therapy (IAVT) can be effective in relieving proximal symptomatic PHCV, only IAVT is a viable treatment option for distal vasospasm. The main advantage of TBA is its long-lasting therapeutic effect and the very low rate of retreatment. However, its use has been associated with a significant risk of serious complications, particularly vessel rupture and reperfusion hemorrhage. Conversely, IAVT is generally considered an effective and low-risk procedure, despite the transient nature of its therapeutic effects and the risk of intracranial hypertension associated with its use. Moreover, newer vasodilator agents appear to have a longer duration of action and a much better safety profile than papaverine, which is rarely used in current clinical practice. Although endovascular treatment of PHCV has been reported to be effective in clinical series, whether it ultimately improves patient outcomes has yet to be demonstrated in a randomized controlled trial.

Role of bilirubin oxidation products in the pathophysiology of DIND following SAH.

Despite intensive research efforts, by our own team and many others, the molecules responsible for acute neurological damage following subarachnoid hemorrhage (SAH) and contributing to delayed ischemic neurological deficit (DIND) have not yet been elucidated. While there are a number of candidate mechanisms, including nitric oxide (NO) scavenging, endothelin-1, protein kinase C (PKC) activation, and rho kinase activation, to name but a few, that have been investigated using animal models and human trials, we are, it seems, no closer to discovering the true nature of this complex and enigmatic pathology. Efforts in our laboratory have focused on the chemical milieu present in hemorrhagic cerebrospinal fluid (CSF) following SAH and the interaction of the environment with the molecules generated by SAH and subsequent events, including NO scavenging, immune response, and clot breakdown. We have identified and characterized a group of molecules formed by the oxidative degradation of bilirubin (a clot breakdown product) and known as BOXes (bilirubin oxidation products). We present a synopsis of the characterization of BOXes as found in human SAH patients' CSF and the multiple signaling pathways by which BOXes act. In summary, BOXes are likely to play an essential role in the etiology of acute brain injury following SAH, as well as DIND.

Cyclocreatine treatment improves cognition in mice with creatine transporter deficiency.

The second-largest cause of X-linked mental retardation is a deficiency in creatine transporter (CRT; encoded by SLC6A8), which leads to speech and language disorders with severe cognitive impairment. This syndrome, caused by the absence of creatine in the brain, is currently untreatable because CRT is required for creatine entry into brain cells. Here, we developed a brain-specific Slc6a8 knockout mouse (Slc6a8-/y) as an animal model of human CRT deficiency in order to explore potential therapies for this syndrome. The phenotype of the Slc6a8-/y mouse was comparable to that of human patients. We successfully treated the Slc6a8-/y mice with the creatine analog cyclocreatine. Brain cyclocreatine and cyclocreatine phosphate were detected after 9 weeks of cyclocreatine treatment in Slc6a8-/y mice, in contrast to the same mice treated with creatine or placebo. Cyclocreatine-treated Slc6a8-/y mice also exhibited a profound improvement in cognitive abilities, as seen with novel object recognition as well as spatial learning and memory tests. Thus, cyclocreatine appears promising as a potential therapy for CRT deficiency.

Ultrasound-enhanced rt-PA thrombolysis in an ex vivo porcine carotid artery model.

Ultrasound is known to enhance recombinant tissue plasminogen activator (rt-PA) thrombolysis. In this study, occlusive porcine whole blood clots were placed in flowing plasma within living porcine carotid arteries. Ultrasonically induced stable cavitation was investigated as an adjuvant to rt-PA thrombolysis. Aged, retracted clots were exposed to plasma alone, plasma containing rt-PA (7.1 ± 3.8 µg/mL) or plasma with rt-PA and Definity® ultrasound contrast agent (0.79 ± 0.47 µL/mL) with and without 120-kHz continuous wave ultrasound at a peak-to-peak pressure amplitude of 0.44 MPa. An insonation scheme was formulated to promote and maximize stable cavitation activity by incorporating ultrasound quiescent periods that allowed for the inflow of Definity®-rich plasma. Cavitation was measured with a passive acoustic detector throughout thrombolytic treatment. Thrombolytic efficacy was measured by comparing clot mass before and after treatment. Average mass loss for clots exposed to rt-PA and Definity® without ultrasound (n = 7) was 34%, and with ultrasound (n = 6) was 83%, which constituted a significant difference (p < 0.0001). Without Definity® there was no thrombolytic enhancement by ultrasound exposure alone at this pressure amplitude (n = 5, p < 0.0001). In the low-oxygen environment of the ischemic artery, significant loss of endothelium occurred but no correlation was observed between arterial tissue damage and treatment type. Acoustic stable cavitation nucleated by an infusion of Definity® enhances rt-PA thrombolysis without apparent treatment-related damage in this ex vivo porcine carotid artery model.

Bilirubin oxidation products seen post subarachnoid hemorrhage have greater effects on aged rat brain compared to young.

INTRODUCTION: We have previously shown that novel oxidation products of Bilirubin, called Bilirubin oxidation products (BOXes), are found in humans and animal models post subarachnoid hemorrhage. We have also proposed that BOXes may play a role in the pathogenesis and clinical complications post SAH. In this study we report on the direct toxicity effects of BOXes on rat brain. METHODS: Identical volumes of either vehicle (normal saline) or BOXes (30 µl of a 20 µM solution) were applied above the dura through a cranial window of young (approximately 7-13 weeks) and aged (approximately 12-18 months) adult male Sprague Dawley rats (Charles River, Wilmington, MA, USA). To determine the extent of BOX-mediated injury, histology and immunocytochemistry were performed at 1, 2, 4, and 7 days post-surgical application of BOXes. We assessed the area of stress gene induction of HSP25/27 and HSP32. Immunohistochemistry was performed using standard avidin-biotin techniques. A monoclonal antibody to HSP25/27 (StressGen, Victoria, British Columbia, Canada), a monoclonal antibody to HSP32/HO-1 (StressGen), and a polyclonal HSP 32/HO-1 antibody were used for the immunocytochemistry. RESULTS: A single dose of BOXes produced substantial increases in HSP25 and HO-1 in the aged rats at all early time points (≤4 days). After 7 days all groups were not significantly different than saline control. Young rats were resistant to BOXes effects compared to saline control with trends towards increased stress gene expression caused by BOXes that did not reach statistical significance. CONCLUSION: We conclude from these studies that BOXes have direct effects on stress gene expression of the cortex post single dose application and that this can be seen for several days with apparent resolution at about 7 days. If BOXes are produced at similar levels in patients, the latency and duration of some SAH complications are consistent with these results.

Do current animal models of intracerebral hemorrhage mirror the human pathology?

Although intracerebral hemorrhage (ICH) has no proven treatment, well-designed studies using animal models of ICH may lead to the development of novel therapies. We briefly review current animal models of ICH. Furthermore, we discuss how these models may be utilized and targeted to facilitate translation of preclinical findings to the clinical arena.

Ultrasound-enhanced delivery of targeted echogenic liposomes in a novel ex vivo mouse aorta model.

The goal of this study was to determine whether targeted, Rhodamine-labeled echogenic liposomes (Rh-ELIP) containing nanobubbles could be delivered to the arterial wall, and whether 1-MHz continuous wave ultrasound would enhance this delivery profile. Aortae excised from apolipoprotein-E-deficient (n=8) and wild-type (n=8) mice were mounted in a pulsatile flow system through which Rh-ELIP were delivered in a stream of bovine serum albumin. Half the aortae from each group were treated with 1-MHz continuous wave ultrasound at 0.49 MPa peak-to-peak pressure, and half underwent sham exposure. Ultrasound parameters were chosen to promote stable cavitation and avoid inertial cavitation. A broadband hydrophone was used to monitor cavitation activity. After treatment, aortic sections were prepared for histology and analyzed by an individual blinded to treatment conditions. Delivery of Rh-ELIP to the vascular endothelium was observed, and sub-endothelial penetration of Rh-ELIP was present in five of five ultrasound-treated aortae and was absent in those not exposed to ultrasound. However, the degree of penetration in the ultrasound-exposed aortae was variable. There was no evidence of ultrasound-mediated tissue damage in any specimen. Ultrasound-enhanced delivery within the arterial wall was demonstrated in this novel model, which allows quantitative evaluation of therapeutic delivery.

Low cerebrospinal fluid and plasma orexin-A (hypocretin-1) concentrations in combat-related posttraumatic stress disorder.

The hypothalamic neuropeptide, orexin-A has a number of regulatory effects in humans and pre-clinical evidence suggests a link to neuroendocrine systems known to be pathophysiologically related to posttraumatic stress disorder (PTSD). However, there are no reports of central nervous system (CNS) or peripheral orexin-A concentrations in patients with PTSD, or any anxiety disorder. Cerebrospinal fluid (CSF) and plasma levels of orexin-A were serially determined in patients with PTSD and healthy comparison subjects to characterize the relationships between orexin-A (in the CNS and peripheral circulation) and central indices of monoaminergic neurotransmission and to determine the degree to which CNS orexin-A concentrations reflect those in the circulating blood. CSF and plasma samples were obtained serially over a 6-h period in 10 male combat veterans with chronic PTSD and 10 healthy male subjects through an indwelling subarachnoid catheter. Orexin-A concentrations were determined in plasma and CSF and CSF levels of the serotonin metabolite, 5-hydroxyindolacetic acid (5-HIAA), and the dopamine metabolite, homovanillic acid (HVA), were determined over the sampling period. CSF and plasma orexin-A concentrations were significantly lower in the patients with PTSD as compared with healthy comparison subjects at all time points. In addition, CSF orexin-A concentrations strongly and negatively correlated with PTSD severity as measured by the Clinician-Administered PTSD Scale (CAPS) in patients with PTSD. Peripheral and CNS concentrations of orexin-A were correlated in the healthy comparison subjects and peripheral orexin-A also correlated with CNS serotonergic tone. These findings suggest low central and peripheral orexin-A activity in patients with chronic PTSD are related to symptom severity and raise the possibility that orexin-A is part of the pathophysiological mechanisms of combat-related PTSD.

Glutathione peroxidase and subarachnoid hemorrhage: implications for the role of oxidative stress in cerebral vasospasm.

OBJECTIVE: Worldwide, cerebral vasospasm after subarachnoid hemorrhage (SAH) has an estimated morbidity and mortality of 1.2 million annually. While it has long been suspected that reactive oxygen species play a major role in the etiology of cerebral vasospasm after SAH, promising results in animal work were not borne out in human clinical trials, despite intensive research effort. The purpose of this study is to investigate the role of glutathione peroxidase in the SAH cerebrospinal fluid milieu. METHODS: We utilized commercially available kits for the quantitation of glutathione peroxidase 1 (glutathione peroxidase) activity and oxygen radical capacity and sodium dodecyl sulfate polyacrylamide gel electrophoresis with Western blotting with specific antibodies to human glutathione peroxidase to determine the enzyme content of the cerebrospinal fluid samples. Human cerebrospinal fluid was obtained in an Institutional Review Board-exempt manner for this study in the following groups: control (no SAH), CSF(C) (SAH but no vasospasm on angiography) and CSF(V) (SAH with clinical and angiographic vasospasm). RESULTS: We found that glutathione peroxidase activity is significantly higher in CSF(V) compared with CSF(C), and this is reflected in a higher total oxidative capacity in CSF(V). Despite similar levels of glutathione peroxidase protein, CSF(V) had significantly greater activity than CSF(C). DISCUSSION: These results further elucidate previous research from this laboratory, showing increased oxidative stress in CSF(V) compared with CSF(C). In conclusion, there appears to be increased glutathione peroxidase activity in CSF(V), despite there being increased levels of oxidative stress markers, suggesting overwhelming oxidative stress may play a role in cerebral vasospasm after SAH.

Mechanical reperfusion is associated with post-ischemic hemorrhage in rat brain.

A major complication of recanalization therapy after an acute arterial occlusion in brain is hemorrhagic transformation (HT). Although it is known that prolonged ischemia is important in the development of HT, the role of reperfusion in ischemia-reperfusion induced HT is less well studied. To address the effect of reperfusion on HT, we assessed the incidence and severity of hemorrhage in rats after 5 h of middle cerebral artery occlusion (MCAO) followed by 19-hour reperfusion compared to rats with permanent occlusion (PMCAO) at the same 24-hour time point. The incidence and amount of hemorrhage, neurological function, and mortality rates were measured. MCAO (5 h) with 19-hour reperfusion was associated with a significantly higher incidence of cortical hemorrhage compared to PMCAO (81.8% vs 18.2%, p<0.05). Hemorrhage scores were higher in the 5-hour MCAO/reperfusion group compared to PMCAO rats (17.6+/-11.5 vs 2.4+/-5.3 in cortex, 20.4+/-4.6 vs 9.7+/-4.5 in striatum, p<0.01). Neurological function was worse in the ischemia-reperfusion group compared to PMCAO (p<0.05) and mortality rates were insignificantly higher in the 5-hour MCAO/reperfusion group vs PMCAO group (54.5% vs 18.1%; p<0.08). The results suggest that reperfusion after prolonged ischemia is associated with increased hemorrhagic transformation and neurological deterioration as compared to permanent ischemia. Whether pharmacological treatments prior to reperfusion attenuate post-ischemic HT requires further study.

Dopamine D2-receptor-mediated increase in vascular and endothelial NOS activity ameliorates cerebral vasospasm after subarachnoid hemorrhage in vitro.

INTRODUCTION: Cerebral vasospasm after subarachnoid hemorrhage (SAH) is a serious complication resulting in delayed neurological deficit, increased morbidity, mortality, longer hospital stays, and rehabilitation time. It afflicts approximately 35 per 100,000 Americans per year, and there is currently no effective therapy. We present in vitro data suggesting that increasing intrinsic nitric oxide relaxation pathways in vascular smooth muscle via dopaminergic agonism ameliorates cerebral vasospasm after SAH. METHODS: Cerebrospinal fluid (CSF) from patients with cerebral vasospasm after SAH (CSF(V)) was used to induce vasospasm in porcine carotid artery in vitro. Dopamine was added to test its ability to reverse spasm, and specific dopamine receptor antagonists were used to determine which receptor mediated the protection. Immunohistochemical techniques confirmed the presence of dopamine receptor subtypes and the involvement of NOS in the mechanism of dopamine protection. RESULTS: Dopamine receptor 1, 2, and 3 subtypes are all present in porcine carotid artery. Dopamine significantly reversed spasm in vitro (67% relaxation), and this relaxation was prevented by Haloperidol, a D(2)R antagonist (10% relaxation, P < 0.05), but not by D(1) or D(3)-receptor antagonism. Both eNOS and iNOS expression were increased significantly in response to CSF(V) alone, and this was significantly enhanced by addition of dopamine, and blocked by Haloperidol. CONCLUSION: Cerebral vasospasm is significantly reversed in a functional measure of vasospasm in vitro by dopamine, via a D(2)R-mediated pathway. The increase in NOS protein seen in both the endothelium and vascular smooth muscle in response to CSF(V) is enhanced by dopamine, also in a D(2)R-dependent mechanism.

Reperfusion activates metalloproteinases that contribute to neurovascular injury.

In this study, we examine the effects of reperfusion on the activation of matrix metalloproteinase (MMP) and assess the relationship between MMP activation during reperfusion and neurovascular injury. Ischemia was produced using suture-induced middle cerebral artery occlusion in rats. The MMP activation was examined with in situ and gel zymography. Injury to cerebral endothelial cells and basal lamina was assessed using endothelial barrier antigen (EBA) and collagen IV immunohistochemistry. Injury to neurons and glial cells was assessed using Cresyl violet staining. These were examined at 3 h after reperfusion (8 h after initiation of ischemia) and compared with permanent ischemia at the same time points to assess the effects of reperfusion. A broad-spectrum MMP inhibitor, AHA (p-aminobenzoyl-Gly-Pro-D-Leu-D-Ala-hydroxamate, 50 mg/kg intravenously) was administered 30 min before reperfusion to assess the roles of MMPs in activating gelatinolytic enzymes and in reperfusion-induced injury. We found that reperfusion accelerated and potentiated MMP-9 and MMP-2 activation and injury to EBA and collagen IV immunopositive microvasculature and to neurons and glial cells in ischemic cortex and striatum relative to permanent ischemia. Administering AHA 30 min before reperfusion decreased MMP-9 activation and neurovascular injury in ischemic cerebral cortex.

PKC and Rho in vascular smooth muscle: activation by BOXes and SAH CSF.

Cerebral vasospasm (CV) remains a significant cause of delayed neurological deficit and ischemic damage after subarachnoid hemorrhage (SAH), despite intensive research effort. The current lack of an effective therapeutic approach is somewhat due to our lack of understanding regarding the mechanism by which this pathological constriction develops. Recent evidence implicates bilirubin oxidation products (BOXes) in the etiology of CV after SAH: BOXes are found in cerebrospinal fluid from SAH patients with symptomatic or angiographically visible vasospasm (CSFV) but not in CSF from SAH patients with no vasospasm (CSFC). We have previously published research suggesting that the etiology of CV comprises two components: a physiological stimulation to constrict and a pathological failure to relax. Both these components are elicited by CSFV, but not CSFC, and BOXes synthesized in the laboratory potentiate physiological constriction in arterial smooth muscle in vitro, and elicit contraction in pial arteries in vivo. In this paper, we will present our results concerning the action of BOXes on arterial smooth muscle constriction, compared with CSFV. We will also present evidence implicating temporal changes in PKC isoforms and Rho expression in both BOXes- and CSFV-elicited smooth muscle responses.

Chemical and biochemical oxidations in spinal fluid after subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a stroke with high rates of mortality and morbidity. SAH-induced cerebral vasospasm can lead to ischemic injury or death and is a common complication of SAH. Recently there has been an accumulation of emerging evidence that oxidation of heme-derived bilirubin into bilirubin oxidation products (BOXes) may be involved in cerebral vasospasm. BOXes are produced by the oxidation of bilirubin yielding a mixture of isomers: 4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide (BOX A) and 3-methyl-5-oxo-4-vinyl- (1,5-dihydropyrrol-2-ylidene)acetamide (BOX B). BOXes have been a subject of interest in the neurosurgical and neurological fields for several years because of their purported correlation with and or role in subarachnoid hemorrhage induced cerebral vasospasm. We believe that it is critical to understand the chemical and biochemical environment in the hemorrhagic spinal fluid after SAH that leads to the oxidation of bilirubin. There is a growing body of information concerning their putative role in vasospasm; however, there is a dearth of information concerning the chemical and biochemical characteristics of BOXes.

An in vitro model of aneurysmal subarachnoid hemorrhage: oxidation of unconjugated bilirubin by cytochrome oxidase.

Aneurysmal subarachnoid hemorrhage is a stroke subtype with high rates of mortality and morbidity. Cerebral vasospasm can lead to ischemic injury or death and is a common complication of aneurysmal subarachnoid hemorrhage, usually occurring 3-9 days afterwards. The cause of vasospasm is not known. Recently, there has been strong evidence that vasoactive oxidation products of bilirubin may be involved. Currently, the factors that lead to bilirubin oxidation are poorly characterized. In this study, we have designed an in vitro model of hemorrhagic stroke in order to investigate conditions that promote the oxidation of bilirubin to form vasoactive compounds. Using our model, we created a basic hematoma system of blood, CSF, and hemeoxygenase-1. We manipulated this system in various ways, incubated it and determined the concentration of vasoactive bilirubin oxidation products that resulted. Conditions where cytochrome oxidase was stimulated caused an increase bilirubin oxidation products (292.6 +/- 39.9 micromol/L respectively, vs. 79.3 +/- 1.3 micromol/L for the basic reaction, p < 0.05), which was attenuated by cyanide. Our data suggest that bilirubin oxidation products may be produced by oxidation(s) requiring an oxygen-utilizing enzyme like cytochrome oxidase.

Expression and function of COOH-terminal myosin heavy chain isoforms in mouse smooth muscle.

Isoforms of the smooth muscle myosin motor, SM1 and SM2, differ in length at the carboxy terminal tail region. Their proportion changes with development, hormonal status and disease, but their function is unknown. We developed mice carrying the myosin heavy chain (MyHC) transgenes SM1, cMyc-tagged SM1, SM2, and V5-tagged SM2, and all transgenes corresponded to the SMa NH(2)-terminal isoform. Transgene expression was targeted to smooth muscle by the smooth muscle alpha-actin promoter. Immunoblot analysis showed substantial expression of the cMyc-tagged SM1 and V5-tagged SM2 MyHC protein in aorta and bladder and transgene mRNA was expressed in mice carrying unlabeled SM1 or SM2 transgenes. Despite significant protein expression of tagged MyHCs we found only small changes in the SM1:SM2 protein ratio. Significant changes in functional phenotype were observed in mice carrying unlabeled SM1 or SM2 transgenes. Force in aorta and bladder was increased (72 +/- 14%, 92 +/- 11%) in SM1 and decreased to 57 +/- 1% and 80 +/- 3% in SM2 transgenic mice. SM1 transgenic bladders had faster (1.8 +/- 0.3 s) and SM2 slower (7.1 +/- 0.5 s) rates of force redevelopment following a rapid step shortening. We hypothesize that small changes in the SM1:SM2 ratio could be amplified if they are associated with changes in thick filament assembly and underlie the altered contractility. These data provide evidence indicating an in vivo function for the COOH-terminal isoforms of smooth muscle myosin and suggest that the SM1:SM2 ratio is tightly regulated in smooth muscle tissues.

Spectrophotometric quantification of bilirubin in hemorrhagic spinal fluid using an innovative algorithm.

Annually, approximately 30,000 people suffer from aneurysmal subarachnoid hemorrhage (SAH) in the United States. In an estimated 5% of these patients, the hemorrhage is difficult to diagnose using conventional methods. Clinicians must rely upon a combination of clinical history, Computerized Tomography (CT) scan evidence and lumbar puncture results to diagnose and differentiate SAH from a traumatic spinal tap (blood in the spinal fluid due to the procedure). Here we describe an algorithm based development of an analytic methodology using visible spectroscopy to reliably quantify bilirubin in hemorrhagic spinal fluid. The analysis, which may be useful for diagnoses concerning hemorrhagic stroke, is based on the detection of bilirubin, and concomitant blood products produced within the Cerebral Spinal Fluid (CSF) following SAH. The algorithm quantifies bilirubin (0.3 to 10 mg/dL) from the resultant absorption spectrum. A model is developed from standard visible spectroscopic absorption curves of bilirubin and hemoglobin by applying traditional Beer's Law principles. The model is coupled to a modified partial least square analysis and control theory concept where the bilirubin is the "signal" and is masked by hemoglobin "noise." This paper describes the computational methods, sensitivity and utility of a system to quantify bilirubin in CSF like solutions containing hemoglobin and bilirubin over 0.5 g/dL-10 g/dL of hemoglobin concentrations.