Spatiotemporal dynamics of pial collateral blood flow following permanent middle cerebral artery occlusion in a rat model of sensory-based protection: a Doppler optical coherence tomography study.

There is a growing recognition regarding the importance of pial collateral flow in the protection from impending ischemic stroke both in preclinical and clinical studies. Collateral flow is also a major player in sensory stimulation-based protection from impending ischemic stroke. Doppler optical coherence tomography has been employed to image spatiotemporal patterns of collateral flow within the dorsal branches of the middle cerebral artery (MCA) as it provides a powerful tool for quantitative in vivo flow parameters imaging (velocity, flux, direction of flow, and radius of imaged branches). It was employed prior to and following dorsal permanent MCA occlusion (pMCAo) in rat models of treatment by protective sensory stimulation, untreated controls, or sham surgery controls. Unexpectedly, following pMCAo in the majority of subjects, some MCA branches continued to show anterograde blood flow patterns over time despite severing of the MCA. Further, in the presence of protective sensory stimulation, the anterograde velocity and flux were stronger and lasted longer than in retrograde flow branches, even within different branches of single subjects, but stimulated retrograde branches showed stronger flow parameters at 24 h. Our study suggests that the spatiotemporal patterns of collateral-based dorsal MCA flow are dynamic and provide a detailed description on the differential effects of protective sensory stimulation.

Hypertension prevents a sensory stimulation-based collateral therapeutic from protecting the cortex from impending ischemic stroke damage in a spontaneously hypersensitive rat model.

Assessing potential stroke treatments in the presence of risk factors can improve screening of treatments prior to clinical trials and is important in testing the efficacy of treatments in different patient populations. Here, we test our noninvasive, nonpharmacological sensory stimulation treatment in the presence of the main risk factor for ischemic stroke, hypertension. Utilizing functional imaging, blood flow imaging, and histology, we assessed spontaneously hypertensive rats (SHRs) pre- and post-permanent middle cerebral artery occlusion (pMCAO). Experimental groups included a treatment SHR group (sensory-stimulated group), control untreated SHR group (no sensory stimulation), and a treated (sensory-stimulated) Wistar-Kyoto normotensive group. Unlike our previous studies, which showed sensory-based complete protection from impending ischemic cortical stroke damage in rats as seen in the treated Wistar-Kyoto group, we found that SHRs at 24hr post-pMCAO lacked evoked cortical activation, had a significant reduction in blood flow within the MCA, and sustained very large infarcts regardless of whether they received stimulation treatment. If translatable, this work highlights a potential need for a combined treatment plan when delivering sensory stimulation treatment in this patient population.

Testing the effects of sensory stimulation as a collateral-based therapeutic for ischemic stroke in C57BL/6J and CD1 mouse strains.

Utilizing a rat model of ischemic stroke, we have previously shown that sensory stimulation can completely protect rats from impending ischemic damage of cortex if this treatment is delivered within the first two hours post-permanent middle cerebral artery occlusion (pMCAo). The current study sought to extend our findings in rats to mice, which would allow new avenues of research not available in rats. Thus, young adult C57BL/6J and CD1 mice were tested for protection from ischemic stroke with the same protective sensory stimulation-based treatment. Cortical activity and blood flow were assessed with intrinsic signal optical imaging (ISOI) and laser speckle imaging (LSI), respectively, and histological analysis (TTC) was performed at the completion of the experiments. Standing in stark contrast to the positive results observed in rats, in both strains we found that there were no differences between treated and untreated mice at 24 hours post-pMCAo in terms of infarct volume, negative functional imaging results, and major reduction in retrograde collateral blood flow as compared to pre-pMCAo baseline and surgical controls. Also, no differences were found between the strains in terms of theses variables. Potential reasons for the differences between rats and mice are discussed.

Fully distributed absolute blood flow velocity measurement for middle cerebral arteries using Doppler optical coherence tomography.

Doppler optical coherence tomography (DOCT) is considered one of the most promising functional imaging modalities for neuro biology research and has demonstrated the ability to quantify cerebral blood flow velocity at a high accuracy. However, the measurement of total absolute blood flow velocity (BFV) of major cerebral arteries is still a difficult problem since it is related to vessel geometry. In this paper, we present a volumetric vessel reconstruction approach that is capable of measuring the absolute BFV distributed along the entire middle cerebral artery (MCA) within a large field-of-view. The Doppler angle at each point of the MCA, representing the vessel geometry, is derived analytically by localizing the artery from pure DOCT images through vessel segmentation and skeletonization. Our approach could achieve automatic quantification of the fully distributed absolute BFV across different vessel branches. Experiments on rodents using swept-source optical coherence tomography showed that our approach was able to reveal the consequences of permanent MCA occlusion with absolute BFV measurement.

Early stimulation treatment provides complete sensory-induced protection from ischemic stroke under isoflurane anesthesia.

Using a rodent model of ischemia [permanent middle cerebral artery occlusion (pMCAO)], previous studies demonstrated that whisker stimulation treatment completely protects the cortex from impending stroke when initiated within 2 h following pMCAO. When initiated 3 h post-pMCAO, the identical treatment exacerbates stroke damage. Rats in these studies, however, were anesthetised with sodium pentobarbital, whereas human stroke patients are typically awake. To overcome this drawback, our laboratory has begun to use the anesthetic isoflurane, which allows rats to rapidly recover from pMCAO within minutes, to test stimulation treatment in awake rats and to determine whether isoflurane has an effect upon the pMCAO stroke model. We found no difference in infarct volume between pMCAO in untreated controls under either sodium pentobarbital or isoflurane, and the primary finding was that rats that received treatment immediately post-pMCAO maintain cortical function and no stroke damage, whereas rats that received treatment 3 h post-pMCAO exhibited eliminated cortical activity and extensive stroke damage. The only difference between anesthetics was the broad extent of evoked cortical activity observed during both functional imaging and electrophysiological recording, suggesting that the extent of evoked activity evident under isoflurane anesthesia is supported by underlying neuronal activity. Given the high degree of similarity with previous data, we conclude that the pMCAO stroke model is upheld with the use of isoflurane. This study demonstrated that the isoflurane-anesthetised rat pMCAO model can be used for cerebrovascular studies, and allows for highly detailed investigation of potential novel treatments for ischemic stroke using awake, behaving animals.

Sensory Stimulation-Based Complete Protection from Ischemic Stroke Remains Stable at 4 Months Post-Occlusion of MCA.

Previous research from our lab has shown that when using a rodent model of ischemic stroke (permanent middle cerebral artery occlusion), mild sensory stimulation, when delivered within two hours of ischemic onset, completely protects the cortex from impending ischemic stroke damage when assessed 24 hours post-occlusion. However, the long-term stability of this protection remains unclear. Using intrinsic signal optical imaging for assessment of cortical function, laser speckle imaging for assessment of blood flow, a battery of behavioral tests and cresyl violet for histological assessment, the present study examined whether this protection was long-lasting. When assessed 4 months post-occlusion (this length of time being equivalent to 10-15 years in humans), rats receiving sensory stimulation treatment immediately after ischemic onset exhibit normal neuronal and vascular function, and they are behaviorally and histologically equivalent to healthy controls (surgical shams). Thus, the complete neuroprotection due to cortical activation via sensory stimulation remains stable with time. These findings add support to the translational potential of this sensory stimulation-based treatment.

Cerebrospinal fluid biomarkers for Parkinson disease diagnosis and progression.

OBJECTIVE: There is a clear need to develop biomarkers for Parkinson disease (PD) diagnosis, differential diagnosis of Parkinsonian disorders, and monitoring disease progression. We and others have demonstrated that a decrease in DJ-1 and/or α-synuclein in the cerebrospinal fluid (CSF) is a potential index for Parkinson disease diagnosis, but not for PD severity. METHODS: Using highly sensitive and quantitative Luminex assays, we measured total tau, phosphorylated tau, amyloid beta peptide 1-42 (Aß(1-42)), Flt3 ligand, and fractalkine levels in CSF in a large cohort of PD patients at different stages as well as healthy and diseased controls. The utility of these 5 markers was evaluated for disease diagnosis and severity/progression correlation alone, as well as in combination with DJ-1 and α-synuclein. The major results were further validated in an independent cohort of cross-sectional PD patients as well as in PD cases with CSF samples collected longitudinally. RESULTS: The results demonstrated that combinations of these biomarkers could differentiate PD patients not only from normal controls but also from patients with Alzheimer disease (AD) and multiple system atrophy. Particularly, with CSF Flt3 ligand, PD could be clearly differentiated from multiple system atrophy, a disease that overlaps with PD clinically, with excellent sensitivity (99%) and specificity (95%). In addition, we identified CSF fractalkine/Aß(1-42) that positively correlated with PD severity in cross-sectional samples as well as with PD progression in longitudinal samples. INTERPRETATION: We have demonstrated that this panel of 7 CSF proteins could aid in Parkinson disease diagnosis, differential diagnosis, and correlation with disease severity and progression.

Complement 3 and factor h in human cerebrospinal fluid in Parkinson's disease, Alzheimer's disease, and multiple-system atrophy.

Complement activation, a key component of neuroinflammation, has been reported in both Parkinson's disease (PD) and Alzheimer's disease (AD). However, it is unclear whether complement activation and neuroinflammation in general are distinctly different from each another in major neurodegenerative disorders. In the present study, cerebrospinal fluid complement 3 (C3) and factor H (FH) were measured and evaluated together with amyloid-ß(42) (Aß(42)), which in recent investigations was decreased in patients with PD, in particular those with cognitive impairment. The study included 345 participants: 126 patients with PD at various stages with or without cognitive impairment, 50 with AD, and 32 with multiple-system atrophy, and 137 healthy control individuals. In addition to changes in Aß(42) concentrations, there were clear differences in the patterns of complement profiles among neurodegenerative disorders. The C3/FH ratio demonstrated high sensitivity and specificity in differentiating patients with multiple-system atrophy from those with AD or PD and control individuals. In addition, the C3/Aß(42) and FH/Aß(42) ratios not only correlated with PD severity approximated using the Unified Parkinson's Disease Rating Scale but also with the presence of cognitive impairment or dementia in PD. Both C3 and FH correlated with the severity of impairment in AD as indicated using Mini-Mental State Examination scores.

Significance and confounders of peripheral DJ-1 and alpha-synuclein in Parkinson's disease.

DJ-1 and alpha-synuclein are leading biomarkers for Parkinson's disease diagnosis and/or monitoring disease progression. A few recent investigations have determined DJ-1 and alpha-synuclein levels in plasma or serum, a more convenient sample source than cerebrospinal fluid; but the results were variable or even contradictory. Besides limitations in detection technology and limited number of cases in some studies, inadequate control of several important confounders likely has contributed to these inconsistent results. In this study, the relative contribution of each blood component to blood DJ-1 and alpha-synuclein was evaluated, followed by quantification of plasma levels of both markers in a larger cohort of patients/subjects ( approximately 300 cases) whose cerebrospinal fluid DJ-1 and alpha-synuclein levels have been determined recently. The results demonstrated that the DJ-1 and alpha-synuclein in blood resided predominantly in red blood cells (>95%), followed by platelets (1-4%), white blood cells and plasma (< or =1%), indicating that variations in hemolysis and/or platelet contamination could have a significant effect on plasma/serum DJ-1 and alpha-synuclein levels. Nonetheless, after adjusting for the age, although there was a trend of decrease in DJ-1 and alpha-synuclein in patients with Parkinson's or Alzheimer's disease compared with healthy controls, no statistical difference was observed in this cohort between any groups, even when the extent of hemolysis and platelet contamination were controlled for. Additionally, no correlation between DJ-1 or alpha-synuclein and Parkinson's disease severity was identified. In conclusion, unlike in cerebrospinal fluid, total DJ-1 or alpha-synuclein in plasma alone is not useful as biomarkers for Parkinson's disease diagnosis or progression/severity.

DJ-1 and alpha-synuclein in human cerebrospinal fluid as biomarkers of Parkinson's disease.

Biomarkers are urgently needed for the diagnosis and monitoring of disease progression in Parkinson's disease. Both DJ-1 and alpha-synuclein, two proteins critically involved in Parkinson's disease pathogenesis, have been tested as disease biomarkers in several recent studies with inconsistent results. These have been largely due to variation in the protein species detected by different antibodies, limited numbers of patients in some studies, or inadequate control of several important variables. In this study, the nature of DJ-1 and alpha-synuclein in human cerebrospinal fluid was studied by a combination of western blotting, gel filtration and mass spectrometry. Sensitive and quantitative Luminex assays detecting most, if not all, species of DJ-1 and alpha-synuclein in human cerebrospinal fluid were established. Cerebrospinal fluid concentrations of DJ-1 and alpha-synuclein from 117 patients with Parkinson's disease, 132 healthy individuals and 50 patients with Alzheimer's disease were analysed using newly developed, highly sensitive Luminex technology while controlling for several major confounders. A total of 299 individuals and 389 samples were analysed. The results showed that cerebrospinal fluid DJ-1 and alpha-synuclein levels were dependent on age and influenced by the extent of blood contamination in cerebrospinal fluid. Both DJ-1 and alpha-synuclein levels were decreased in Parkinson's patients versus controls or Alzheimer's patients when blood contamination was controlled for. In the population aged > or = 65 years, when cut-off values of 40 and 0.5 ng/ml were chosen for DJ-1 and alpha-synuclein, respectively, the sensitivity and specificity for patients with Parkinson's disease versus controls were 90 and 70% for DJ-1, and 92 and 58% for alpha-synuclein. A combination of the two markers did not enhance the test performance. There was no association between DJ-1 or alpha-synuclein and the severity of Parkinson's disease. Taken together, this represents the largest scale study for DJ-1 or alpha-synuclein in human cerebrospinal fluid so far, while using newly established sensitive Luminex assays, with controls for multiple variables. We have demonstrated that total DJ-1 and alpha-synuclein in human cerebrospinal fluid are helpful diagnostic markers for Parkinson's disease, if variables such as blood contamination and age are taken into consideration.

Glycoproteomics in neurodegenerative diseases.

Protein glycosylation regulates protein function and cellular distribution. Additionally, aberrant protein glycosylations have been recognized to play major roles in human disorders, including neurodegenerative diseases. Glycoproteomics, a branch of proteomics that catalogs and quantifies glycoproteins, provides a powerful means to systematically profile the glycopeptides or glycoproteins of a complex mixture that are highly enriched in body fluids, and therefore, carry great potential to be diagnostic and/or prognostic markers. Application of this mass spectrometry-based technology to the study of neurodegenerative disorders (e.g., Alzheimer's disease and Parkinson's disease) is relatively new, and is expected to provide insight into the biochemical pathogenesis of neurodegeneration, as well as biomarker discovery. In this review, we have summarized the current understanding of glycoproteins in biology and neurodegenerative disease, and have discussed existing proteomic technologies that are utilized to characterize glycoproteins. Some of the ongoing studies, where glycoproteins isolated from cerebrospinal fluid and human brain are being characterized in Parkinson's disease at different stages versus controls, are presented, along with future applications of targeted validation of brain specific glycoproteins in body fluids.