Personalizing non-small cell lung cancer treatment through patient-derived xenograft models: preclinical and clinical factors for consideration.

PURPOSE: In the pursuit of creating personalized and more effective treatment strategies for lung cancer patients, Patient-Derived Xenografts (PDXs) have been introduced as preclinical platforms that can recapitulate the specific patient's tumor in an in vivo model. We investigated how well PDX models can preserve the tumor's clinical and molecular characteristics across different generations. METHODS: A Non-Small Cell Lung Cancer (NSCLC) PDX model was established in NSG-SGM3 mice and clinical and preclinical factors were assessed throughout subsequent passages. Our cohort consisted of 40 NSCLC patients, which were used to create 20 patient-specific PDX models in NSG-SGM3 mice. Histopathological staining and Whole Exome Sequencing (WES) analysis were preformed to understand tumor heterogeneity throughout serial passages. RESULTS: The main factors that contributed to the growth of the engrafted PDX in mice were a higher grade or stage of disease, in contrast to the long duration of chemotherapy treatment, which was negatively correlated with PDX propagation. Successful PDX growth was also linked to poorer prognosis and overall survival, while growth pattern variability was affected by the tumor aggressiveness, primarily affecting the first passage. Pathology analysis showed preservation of the histological type and grade; however, WES analysis revealed genomic instability in advanced passages, leading to the inconsistencies in clinically relevant alterations between the PDXs and biopsies. CONCLUSIONS: Our study highlights the impact of multiple clinical and preclinical factors on the engraftment success, growth kinetics, and tumor stability of patient-specific NSCLC PDXs, and underscores the importance of considering these factors when guiding and evaluating prolonged personalized treatment studies for NSCLC patients in these models, as well as signaling the imperative for additional investigations to determine the full clinical potential of this technique.

Visualization of both proximal M2-MCA segments in patients (the Tilted-V Sign) with acute M1-MCA occlusion stroke is associated with better procedural and prognostic outcomes.

Introduction: We aimed to assess the clinical significance of M1-MCA occlusion with visualization of both MCA-M2 segments ["Tilted-V sign" (TVS)] on initial CT angiography (CTA) in patients with acute ischemic stroke (AIS) undergoing endovascular thrombectomy (EVT). Methods: Data for patients with consecutive AIS undergoing EVT for large vessel occlusion (LVO) in two academic centers are recorded in ongoing databases. Patients who underwent EVT for M1-MCA occlusions ≤ 6 h from symptom onset were included in this retrospective analysis. Results: A total of 346 patients met the inclusion criteria; 189 (55%) had positive TVS. Patients with positive TVS were younger (68 ± 14 vs. 71 ± 14 years, P = 0.028), with similar rates of vascular risk factors and baseline modified Rankin scores (mRS) 0-2. The rates of achieving thrombolysis in cerebral ischemia (TICI) 2b-3 were similar to the two groups (79%), although successful first-pass recanalization was more common with TVS (64 vs. 36%, p = 0.01). On multivariate analysis, higher collateral score [odds ratio (OR) 1.38 per unit increase, p = 0.008] and lower age (OR 0.98 per year increase, p = 0.046) were significant predictors of TVS. Patients with positive TVS had higher post-procedural Alberta Stroke Program Early CT Score (ASPECTS; 6.9 ± 2.2 vs. 5.2 ± 2.3, p = 0.001), were discharged with lower National Institutes of Health Stroke Score (NIHSS; 6±6 vs. 9±7, p = 0.003) and higher rates of mRS 0-2 (29.5 vs. 12%, p = 0.001), and had lower rates of 90-day mortality (13.2 vs. 21.6%, p = 0.038). However, TVS was not an independent predictor of functional independence (OR 2.51; 95% CI 0.7-8.3). Conclusion: Tilted-V Sign, an easily identifiable radiological marker, is associated with fewer recanalization attempts, better functional outcomes, and reduced mortality.

Frequency- and spike-timing-dependent mitochondrial Ca2+ signaling regulates the metabolic rate and synaptic efficacy in cortical neurons.

Mitochondrial activity is crucial for the plasticity of central synapses, but how the firing pattern of pre- and postsynaptic neurons affects the mitochondria remains elusive. We recorded changes in the fluorescence of cytosolic and mitochondrial Ca2+ indicators in cell bodies, axons, and dendrites of cortical pyramidal neurons in mouse brain slices while evoking pre- and postsynaptic spikes. Postsynaptic spike firing elicited fast mitochondrial Ca2+ responses that were about threefold larger in the somas and apical dendrites than in basal dendrites and axons. The amplitude of these responses and metabolic activity were extremely sensitive to the firing frequency. Furthermore, while an EPSP alone caused no detectable Ca2+ elevation in the dendritic mitochondria, the coincidence of EPSP with a backpropagating spike produced prominent, highly localized mitochondrial Ca2+ hotspots. Our results indicate that mitochondria decode the spike firing frequency and the Hebbian temporal coincidences into the Ca2+ signals, which are further translated into the metabolic output and most probably lead to long-term changes in synaptic efficacy.

A Novel Histological Technique to Assess Severity of Traumatic Brain Injury in Rodents: Comparisons to Neuroimaging and Neurological Outcomes.

Here we evaluate an alternative protocol to histologically examine blood-brain barrier (BBB) breakdown, brain edema, and lesion volume following traumatic brain injury (TBI) in the same set of rodent brain samples. We further compare this novel histological technique to measurements determined by magnetic resonance imaging (MRI) and a neurological severity score (NSS). Sixty-six rats were randomly assigned to a sham-operated, mild TBI, moderate TBI, or severe TBI group. 48 h after TBI, NSS, MRI and histological techniques were performed to measure TBI severity outcome. Both the histological and MRI techniques were able to detect measurements of severity outcome, but histologically determined outcomes were more sensitive. The two most sensitive techniques for determining the degree of injury following TBI were NSS and histologically determined BBB breakdown. Our results demonstrate that BBB breakdown, brain edema, and lesion volume following TBI can be accurately measured by histological evaluation of the same set of brain samples.

Tumor-Treating Fields for the treatment of glioblastoma: a systematic review and meta-analysis.

BACKGROUND: Tumor-Treating Fields (TTFields) is an emerging treatment modality for glioblastoma (GBM). Studies have shown a good safety profile alongside improved efficacy in newly diagnosed GBM (ndGBM), while a less clear effect was shown for recurrent GBM (rGBM). Despite regulatory support, sectors of the neuro-oncology community have been reluctant to accept it as part of the standard treatment protocol. To establish an objective understanding of TTFields' mechanism of action, safety, efficacy, and economical implications, we conducted a systematic literature review and meta-analysis. METHODS: A systematic search was conducted in PubMed, Scopus, and Cochrane databases. Twenty studies met the pre-defined inclusion criteria, incorporating 1636 patients (542 ndGBM and 1094 rGBM), and 11 558 patients (6403 ndGBM and 5155 rGBM) analyzed for the clinical outcomes and safety endpoints, respectively. RESULTS: This study demonstrated improved clinical efficacy and a good safety profile of TTFields. For ndGBM, pooled median overall survival (OS) and progression-free survival (PFS) were 21.7 (95%CI = 19.6-23.8) and 7.2 (95%CI = 6.1-8.2) months, respectively. For rGBM, pooled median OS and PFS were 10.3 (95%CI = 8.3-12.8) and 5.7 (95%CI = 2.8-10) months, respectively. Compliance of ≥75% was associated with an improved OS and the predominant adverse events were dermatologic, with a pooled prevalence of 38.4% (95%CI = 32.3-44.9). Preclinical studies demonstrated TTFields' diverse molecular mechanism of action, its potential synergistic efficacy, and suggest possible benefits for certain populations. CONCLUSIONS: This study supports the use of TTFields for GBM, alongside the standard-of-care treatment protocol, and provides a practical summary, discussing the current clinical and preclinical aspects of the treatment and their implication on the disease course.

Subcellular distribution of persistent sodium conductance in cortical pyramidal neurons.

Cortical pyramidal neurons possess a persistent Na+ current (INaP) which, in contrast to the larger transient current, does not undergo rapid inactivation. Although relatively quite small, INaP is active at subthreshold voltages and therefore plays an important role in neuronal input-output processing. The subcellular distribution of channels responsible for INaP and the mechanisms which render them persistent are not known. Using high-speed fluorescence Na+ imaging and whole-cell recordings in brain slices obtained from mice of either sex, we reconstructed the INaP elicited by slow voltage ramps in soma and processes of cortical pyramidal neurons. We found that in all neuronal compartments, the relationship between persistent Na+ conductance and membrane voltage has the shape of a Boltzmann function. Although the density of channels underlying INaP was about twofold lower in the axon initial segment (AIS) than in the soma, the axonal channels were activated by about 10 mV less depolarization than were somatic channels. This difference in voltage dependence explains why, at functionally critical subthreshold voltages, most INaP originates in the AIS. Finally, we show that endogenous polyamines constrain INaP availability in both somato-dendritic and axonal compartments of non-dialyzed cortical neurons.SIGNIFICANCE STATEMENT:The most salient characteristic of neuronal sodium channels is fast inactivation. However, a fraction of the sodium current does not inactivate. In cortical neurons, persistent current (INaP) plays a prominent role in many important functions. Its subcellular distribution and generation mechanisms are, however, elusive. Using high-speed fluorescence Na+ imaging and electrical recordings, we reconstructed the INaP in soma and processes of cortical pyramidal neurons. We found that at near-threshold voltages INaP originates predominately from the axon, due to the distinctive voltage dependence of the underlying channels and not because of their high density. Finally, we show that the presence of endogenous polyamines significantly constrains INaP availability in all compartments of non-dialyzed cortical neurons.

A Complex Diving-For-Food Task to Investigate Social Organization and Interactions in Rats.

For many species, where status is a vital motivator that can affect health, social hierarchies influence behavior. Social hierarchies that include dominant-submissive relationships are common in both animal and human societies. These relationships can be affected by interactions with others and with their environment, making them difficult to analyze in a controlled study. Rather than a simple dominance hierarchy, this formation has a complicated presentation that allows rats to avoid aggression. Status can be stagnant or mutable, and results in complex societal stratifications. Here we describe a complex diving-for-food task to investigate rodent social hierarchy and behavioral interactions. This animal model may allow us to assess the relationship between a wide range of mental illnesses and social organization, as well as to study the effectiveness of therapy on social dysfunction.

A Metric Test for Assessing Spatial Working Memory in Adult Rats following Traumatic Brain Injury.

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of human studies, animal models provide suitable alternatives to test treatment methods, and to study the mechanisms and related complications of the condition. Experimental rodent models have historically been the most widely used due to their accessibility, low cost, reproducibility, and validated approaches. A metric test, which tests the ability to recall the placement of two objects at various distances and angles from one another, is a technique to study impairment in spatial working memory (SWM) after TBI. The significant advantages of metric tasks include the possibility of dynamic observation, low cost, reproducibility, relative ease of implementation, and low stress environment. Here, we present a metric test protocol to measure impairment of SWM in adult rats after TBI. This test provides a feasible way to evaluate physiology and pathophysiology of brain function more effectively.

Successful management of spinal cord ischemia in a pediatric patient with fibrocartilaginous embolism: illustrative case.

BACKGROUND: Fibrocartilaginous embolism (FCE) is a rare cause of ischemic myelopathy that occurs when the material of the nucleus pulposus migrates into vessels supplying the spinal cord. The authors presented a case of pediatric FCE that was successfully managed by adapting evidence-based recommendations used for spinal cord neuroprotection in aortic surgery. OBSERVATIONS: A 7-year-old boy presented to the emergency department with acute quadriplegia and hemodynamic instability that quickly progressed to cardiac arrest. After stabilization, the patient regained consciousness but remained in a locked-in state with no spontaneous breathing. The patient presented a diagnostic challenge. Traumatic, inflammatory, infectious, and ischemic etiologies were considered. Eventually, the clinical and radiological findings led to the presumed diagnosis of FCE. Treatment with continuous cerebrospinal fluid drainage (CSFD), pulse steroids, and mean arterial pressure augmentation was applied, with subsequent considerable and consistent neurological improvement. LESSONS: The authors proposed consideration of the adaptation of spinal cord neuroprotection principles used routinely in aortic surgery for the management of traumatic spinal cord ischemia (FCE-related in particular), namely, permissive arterial hypertension and CSFD. This is hypothesized to allow for the maintenance of sufficient spinal cord perfusion until adequate physiological blood perfusion is reestablished (remodeling of the collateral arterial network and/or clearing/absorption of the emboli).

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples.

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

Minimally Invasive Subtemporal Intradural Approach for Penetrating Orbitocranial Injury by Wooden Foreign Body Into the Lateral Wall of the Cavernous Sinus.

Non-missile transorbital penetrating head injuries are relatively rare, though potentially fatal injuries. Trajectory for intracranial entrance is typically via the orbital roof, the superior orbital fissure (SOF), or the optic canal. Non-metallic intracranial penetrating injuries are even scarcer and may pose unusual diagnostic and surgical challenges. Here we present and discuss a unique case of a penetrating injury by a wooden foreign body (FB) which entered and expanded the inter-dural space of the lateral cavernous sinus (CS) sinus wall without intracavernous or intradural involvement. The patient was a 71 year-old male who fell face-down and sustained a penetrating transorbital injury by a dry twig fragment, which passed through the SOF and into the interdural space of lateral wall of the ipsilateral CS. The patient was fully conscious (GCS15) at presentation but had severe ocular injury (complete ophthalmoplegia and blindness of the injured eye). The wooden FB was successfully removed via a minimally invasive subtemporal intradural approach with no apparent immediate or long-term complications. We emphasize the unusual diagnostic and surgical challenges related to this kind of rare injuries as reflected by the decision-making considerations taken in the presented case.

Laser-Induced Brain Injury in the Motor Cortex of Rats.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models. When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed. Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

Single root multiple spinal schwannomas: Case report, treatment strategy and review of literature.

INTRODUCTION: Schwannomatosis is defined as multiple schwannomas without presence of neurofibromatosis and is a rare pathology. In vast majority of cases the schwannomas grow from different nerve roots or peripheral nerves. PRESENTATION OF CASE: A 52-year-old woman presented with multiple intradural schwannomas arranged in a chain along the spinal canal causing significant compression. The lesions were successfully removed using a left side en-bloc hemilaminectomy technique in order to preserve maximal stability of the posterior column. Back and leg pain resolved completely. Tendon reflexes returned to normal shortly. There was decreased pain sensation in the distribution of the left L3 spinal root. DISCUSSION: The traditional surgical strategy for posterior approach by laminectomy or laminotomy is sometimes complicated with instability or deformation of the vertebral column that requires surgical stabilization. We performed a one side en-bloc hemilaminectomy thus maintaining the integrity of the muscles and ligaments on the opposite side and preserving maximal stability of the vertebral column. Densely adherent tumors required careful sharp dissection and separation under neurosurgical monitoring and stimulation for recognition and preservation of spinal roots. An additional tumor was discovered by exploration of the spinal canal using an endoscope. CONCLUSION: Multiple spinal cord schwannomas that are growing along the same part of the vertebral column can be safely removed by one-sided hemilaminectomy with preservation of the integrity of the muscles and ligaments on the opposite side and thus maintain spinal stability. The 30° endoscope can be a good tool for visual exploration of the spinal canal.

The Chiasmal Compression Index: An Integrative Assessment Tool for Visual Disturbances in Patients with Pituitary Macroadenomas.

OBJECTIVE: Visual status is routinely evaluated by neuro-ophthalmologic examination and computerized visual field (VF) tests in patients with chiasmal compression secondary to pituitary macroadenoma. Currently, no relevant data exists to accurately quantify the extent of optic apparatus compromise to further guide clinical decision-making. We aimed to assess for a possible quantitative correlation between optic chiasm geometric properties on magnetic resonance imaging (MRI) and VF deficits. METHODS: Visual assessments and concurrent MRI scans were retrospectively reviewed from patients treated for pituitary macroadenoma in a single medical institution. Chiasm width, chiasm minimal and maximal height, and chiasm angle were measured on MRI coronal plane images by 3 independent reviewers (for the sake of variability analysis). VF numerical summary parameters were also retrieved. RESULTS: A total of 30 patients were included in the final analysis. Average VF index was 70% (±30), and averaged mean deviation was 10.0 db (±9). Chiasm angle and width (which together represents the bending and stretching of the chiasm by the upward directed compression; both of which demonstrated high inter- and intraobserver agreement) showed strong correlation with VF loss. Chiasmal compression index derived from those parameters showed even stronger correlation. CONCLUSIONS: The strong correlation demonstrated by our results of this relatively simple radiologic measurement with VF status, despite the relatively small cohort, calls for further investigation in this promising direction, and may facilitate with basic assessment and clinical decision-making for patients with equivocal neuro-ophthalmologic evaluation, as well as with poor compliance.

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration.

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

Hemoglobin Levels and Red Blood Cells Distribution Width Highlights Glioblastoma Patients Subgroup With Improved Median Overall Survival.

Glioblastoma multiforme (GBM) is known for its dismal prognosis, though its dependence on patients' readily available RBCs parameters is not fully established. In this work, 170 GBM patients, diagnosed and treated in Soroka University Medical Center (SUMC) over the last 12 years were retrospectively inspected for their survival dependency on pre-operative RBCs parameters. Besides KPS and tumor resection supplemented by oncological treatment, age under 70 (HR = 0.4, 95% CI 0.24-0.65, p = 0.00073), low hemoglobin level (HR = 1.79, 95% CI 1.06-2.99, p = 0.031), and Red Cell Distribution Width (RDW) < 14% (HR = 0.57, 95% CI 0.37-0.88, p = 0.018) were found to be prognostic of patients' overall survival in multivariate analysis, accounting for a false discovery rate of < 5% due to multiple hypothesis testing. According to these results, a stratification tree was made, from which a favorable route highlighted a subgroup of nearly 30% of the cohorts' patients whose median overall survival was 21.1 months (95% CI 16.2-27.2)-higher than the established chemo-radiation standard first-line treatment regimen overall median survival average of about 15 months. The beneficial or detrimental effect of RBCs parameters on GBM prognosis and its possible causes is discussed.

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl4) Exposure Through an Orogastric Tube.

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including complications such as hepatic encephalopathy and impaired protein synthesis. Appropriate animal models are vital to test the mechanism and pathophysiology of ALI and investigate different hepatoprotective strategies. Due to its ability to perform chemical transformations, carbon tetrachloride (CCl4) is widely used in the liver to induce ALI through the formation of reactive oxygen species. CCl4 exposure can be performed intraperitoneally, by inhalation, or through a nasogastric or orogastric tube. Here, we describe a rodent model, in which ALI is induced by CCl4 exposure through an orogastric tube. This method is inexpensive, easily performed, and has minimal hazard risk. The model is highly reproducible and can be widely used to determine the efficacy of potential hepatoprotective strategies and assess markers of liver injury.

A Novel Method for Assessing Cerebral Edema, Infarcted Zone and Blood-Brain Barrier Breakdown in a Single Post-stroke Rodent Brain.

Stroke is a major cause of global morbidity and mortality. Middle cerebral artery occlusion (MCAO) has historically been the most common animal model of simulating ischemic stroke. The extent of neurological injury after MCAO is typically measured by cerebral edema, infarct zone, and blood-brain barrier (BBB) permeability. A significant limitation of these methods is that separate sets of brains must be used for each measurement. Here we examine an alternative method of measuring cerebral edema, infarct zone and BBB permeability following MCAO in the same set of brain samples. Ninety-six rats were randomly divided into three experimental groups. Group 1 (n = 27) was used for the evaluation of infarct zone and brain edema in rats post-MCAO (n = 17) vs. sham-operated controls (n = 10). Group 2 (n = 27) was used for the evaluation of BBB breakdown in rats post-MCAO (n = 15) vs. sham-operated controls (n = 10). In Group 3 (n = 42), all three parameters were measured in the same set of brain slices in rats post-MCAO (n = 26) vs. sham-operated controls (n = 16). The effect of Evans blue on the accuracy of measuring infarct zone by 2,3,5-triphenyltetrazolium chloride (TTC) staining was determined by measuring infarct zone with and without an applied blue filter. The effects of various concentrations of TTC (0, 0.05, 0.35, 0.5, 1, and 2%) on the accuracy of measuring BBB permeability was also assessed. There was an increase in infarct volume (p < 0.01), brain edema (p < 0.01) and BBB breakdown (p < 0.01) in rats following MCAO compared to sham-operated controls, whether measured separately or together in the same set of brain samples. Evans blue had an effect on measuring infarct volume that was minimized by the application of a blue filter on scanned brain slices. There was no difference in the Evans blue extravasation index for the brain tissue samples without TTC compared to brain tissue samples incubated in TTC. Our results demonstrate that measuring cerebral edema, infarct zone and BBB permeability following MCAO can accurately be measured in the same set of brain samples.

Unfavorable functional outcome is expected for elderly patients suffering from acute subdural hematoma even when presenting with preserved level of consciousness.

Alongside an increase in life expectancy, median age of patients presenting with traumatic acute subdural hematomas (ASDH) has increased as well. Treatment guidelines are based on studies performed on relatively young patients. The optimal management of elderly (>70 years old) patients with ASDH, specifically those with relatively preserved level of consciousness, was not thoroughly investigated so far. We retrospectively examined elderly patients presented to our medical center between the years 2006-2016 with traumatic convexity ASDH and GCS of 13-15. 773 patients were included in the initial cohort and 54 patients were included in the final analysis. The mean age at presentation was 81.5 years and the means of hematoma thickness and midline shift were 15.5 mm and 6.6 mm, respectively. Patients in our cohort had an overall unfavorable outcome (mRS 5-6) of 28% and 56% at discharge and at 1 year following injury, respectively. The results were not significantly different for the subgroups of patients older than 80 years and patients with high ASA-PS. Surgical evacuation of the ASDH was undertaken in 28 patients with focal neurologic deficit and/or worsening on subsequent brain scans. At 1 year, 64% (18 patients) in the surgery group had unfavorable outcome compared to 48% (12 patients) in the conservative group. We believe that these numbers should be taken under consideration when assessing elderly patients with convexity ASDH and relatively preserved level of consciousness.