Computer-assisted Intravital Microscopy Research Articles

Microvasculopathy and biomarkers in sickle cell disease: the promise of non‐invasive real‐time in vivo tools

Microvasculopathy and biomarkers in sickle cell disease: the promise of non‐invasive real‐time <i>in vivo</i> tools

Homocysteine is associated with severity of microvasculopathy in sickle cell disease patients.

The pathophysiology of sickle cell disease (SCD) includes vasculopathy as well as anaemia. Elevated plasma homocysteine is a risk factor for vascular disease and may be associated with increased risk of vascular complications in SCD patients. In the present study, microvascular characteristics were assessed in the bulbar conjunctiva of 18 paediatric and 18 adult SCD patients, using the non-invasive technique of computer-assisted intravital microscopy. A vasculopathy severity index (SI) was computed to quantify the degree of microvasculopathy in each patient. Plasma homocysteine and several of its determinants [serum folate and vitamin B12, plasma pyridoxal-5'-phosphate (vitamin B6 status) and creatinine (kidney function)] were measured. Age was strongly correlated with microvasculopathy in the SCD patients, with the SI increasing about 0·1 unit per one-year increase in age (P<0·001). After adjusting for age, gender, B-vitamin status and creatinine, homocysteine concentration was directly correlated with severity index (P<0·05). Age and homocysteine concentration were independent predictors of microvasculopathy in SCD patients. It remains to be determined whether lowering homocysteine concentrations using appropriate B-vitamin supplements (folate and vitamins B12 and B6) - particularly if started early in life - could ameliorate microvasculopathy and its associated complications in SCD patients.

Microvascular complications in orthokeratology (Ortho-K): A real-time study on the microvasculature of the bulbar conjunctiva in Ortho-K treatment.

Orthokeratology (Ortho-K) is an over-night hard contact lens therapy, which physically reshapes the corneal curvature in order to stabilize or temporally eliminate myopia in patients. We hypothesize that the prolonged physical contact and mechanical pressure induced by the Ortho-K lenses may create lasting inadvertent effects and damages (microangiopathy), and may bring about unwanted changes in the microvasculature of the bulbar conjunctiva. Computer-assisted intravital microscopy (CAIM) was used to view, document (via videotaping) and objectively quantify (via computer-assisted image analysis) the real-time dynamic and morphometric characteristics of the conjunctival microcirculation in long-term (at least over one year) Ortho-K patients (n = 11) and matched non-user control subjects (n = 8). Ortho-K patients were instructed to wear their lenses overnight following standard protocol. During the study, the conjunctival microcirculation of the left eye of all Ortho-K lens users was viewed, frequently re-focused and videotaped, without and with the lens in place, as outlined in Methods. The matched control subjects (non-lens wearing) were videotaped and studied in like manner. The dynamic and morphometric characteristics of each user and control subject were analyzed, quantified and summated as a severity index (SI) collectively for comparison. SI of Ortho-K lens users (4.18±1.08) differed significantly from SI of control subjects (1.75±1.39, p≤0.05). In addition, changes in the conjunctival microcirculation (e.g., flow velocity, vessel diameter, shape change, etc) were viewed and videotaped immediately after the myopic patients put on the Ortho-K lenses. Eight of the 11 Ortho-K lens users displayed significant percentage changes (p≤0.05) in flow velocity and 10 of 11 displayed significant percentage changes (p≤0.05) in vessel diameter, without and with the Ortho-K lenses. The results clearly indicated that significant microvascular changes via tissue remodeling occurred, and were caused directly by the physical presence of the Ortho-K lenses.

Results of computer-assisted intravital microscopy in overweight and obesity

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Real-time studies of hypertension using non-mydriatic fundus photography and computer-assisted intravital microscopy

Hypertension is asymptomatic until late stages of pathogenesis, rendering an effective means of detection for early diagnosis essential. The current method of diagnosing hypertension requires two or more sphygmomanometric readings over two or more office visits, which potentially hinders early detection. Though retinopathy is an indicator of vascular damage, it generally presents in later stages of hypertension. Previous and related studies have suggested that the microvasculature in the bulbar conjunctiva may be a sensitive site to assess vasculopathy. Conjunctival microangiopathy was assessed using CAIM and reported on a severity index (SI). Images of the retinal fundus were taken via non-mydriatic fundus photography and graded using the Scheie scale in the same subjects to compare with CAIM. Conjunctival microangiopathy was significantly elevated in hypertensive subjects (SI = 5.35 ± 1.04, n = 20) compared to control subjects (SI = 1.75 ± 1.39, n = 8; p ≤ 0.05), and correlated with time since disease diagnosis (R² = 0.33). Hypertensive subjects with Grade 1 retinopathy displayed increased conjunctival microangiopathy (SI = 5.85 ± 0.90, n = 13) compared to those without retinopathy (SI = 4.43 ± 0.53, n = 7; p ≤ 0.05). These data indicate a possible pre-retinopathy time window during which conjunctival microangiopathy may indicate the risk of organ damage, supporting the hypothesis that the conjunctival microcirculation may serve as a platform for early detection and monitoring disease progression.

Monocyte Chemotactic Protein-1 Is Associated with Microvascular Abnormalities and Serum Ferritin Concentrations in Sickle Cell Disease Patients

Abstract 3255 Introduction:Microvascular abnormalities underlie much of the morbidity observed in sickle cell disease (SCD). Adherence of sickle erythrocytes and leukocytes to the microvascular endothelium leads to vaso-occlusion and end organ ischemia/reperfusion injury. Monocyte chemotactic protein-1 (MCP-1) is a chemokine involved in the recruitment of monocytes to sites of endothelial activation or injury and thus contributes to the pathophysiology of vascular disease. In SCD patients, circulating MCP-1 concentrations are elevated during both steady state and vaso-occlusive crisis. However, the relationship between MCP-1 and microvascular abnormalities in SCD patients has not been assessed. In the present study, we measured a panel of circulating cytokines including serum MCP-1 and hematological biomarkers including serum ferritin in SCD patients who were being monitored by computer-assisted intravital microscopy (CAIM) to assess severity of microvasculopathy. Methods:The study included 31 steady state SCD patients (22 females, 9 males) ranging in age from 4–61 years. MCP-1 concentrations were measured by Luminex multiple analyte profiling. Ferritin concentrations were measured by chemiluminometric immunossay. The conjunctival microvasculature was assessed using CAIM. A severity index on a scale of 0–15 was calculated as described previously (Cheung et al, Am J Hematol 85:899, 2010) to quantify the degree of microvasculopathy observed among the patients. Results:The mean ± SD MCP-1 concentration was 445 ± 253 pg/ml (range: 122–1180 pg/ml), mean ± SD ferritin concentration was 1675 ± 1883 ng/ml (range 18–5825 ng/ml), and mean ± SD severity index was 5.0 ± 2.5 (range: 0–9). By multiple regression analysis, with controlling for age and gender, MCP-1 was directly correlated with severity index (p = 0.046), and serum ferritin was directly correlated with MCP-1 (p = 0.019). Conclusions:Monocyte recruitment to the site of endothelial injury involves chemotactic signaling mediated in part by MCP-1, levels of which rise in SCD. Our results indicate that MCP-1 is associated with and may directly contribute to the microvasculopathy observed in SCD patients. Furthermore, we find that MCP-1 levels are correlated with ferritin concentrations. Ferritin reflects body iron stores, and an increase of iron stores in SCD resulting from chronic blood transfusion as well as increased iron absorption associated with hemolysis may thus lead to monocyte/macrophage recruitment to the vascular endothelium and contribute to vasculopathy. Measurements of MCP-1 and ferritin may also serve as surrogate biomarkers of microvasculopathy severity and inflammation. Disclosures:Green:Emisphere - Consultancy : Consultancy; Teva Pharmaceuticals - expert testimony: Consultancy.

Exchange Transfusion Therapy and Its Effects on Real-time Microcirculation in Pediatric Sickle Cell Anemia Patients

Periodic blood exchange transfusion is a treatment modality commonly used to manage pediatric sickle cell anemia at the University of California Davis Medical Center. The goal of exchange transfusion therapy is to ameliorate vasoocclusion and improve tissue perfusion by removing sickled red blood cells and introducing normal red blood cells. Using computer-assisted intravital microscopy, pretransfusion and posttransfusion microvascular characteristics were analyzed. In this study, the bulbar conjunctiva exhibited a "blanched" avascular appearance in all 6 pediatric sickle cell anemia patients before transfusion, indicative of tissue hypoperfusion and ischemia. Immediately after transfusion, substantial improvement in vascularization and tissue perfusion resulted, reflected by the enhanced appearance of capillaries and arterioles. In addition, a decrease in red cell velocity was observed. These observations provide evidence that exchange transfusion therapy is beneficial in ameliorating vasoocclusion and improving tissue perfusion. However, with the paradoxical posttransfusion decrease in red cell velocity presumably due to induced hyperviscosity from the large transfusion volume, blood flow is still impaired. This decreased velocity may thwart efforts to improve oxygen delivery through transfusion and may, to some extent, promote vasoocclusion instead. This paradoxical result warrants further investigation on the effects of transfusion volume and viscosity in the exchange transfusion process.

Exchange transfusion therapy and its effects on real‐time microcirculation in pediatric sickle cell anemia patients

The goal of exchange transfusion therapy is to ameliorate vasoocclusion and improve tissue perfusion by removing sickled red blood cells and introducing normal red blood cells. Using computer‐assisted intravital microscopy, pre‐transfusion and post‐transfusion microvascular characteristics were analyzed and compared. In this study, the bulbar conjunctiva exhibited a “blanched” avascular appearance in all 6 pediatric sickle cell anemia patients before transfusion, indicative of tissue hypoperfusion and ischemia. Immediately after transfusion, substantial improvement in vascularization and tissue perfusion resulted. In addition, a decrease in red cell velocity was observed. These observations provide evidence that exchange transfusion therapy is beneficial in ameliorating vasoocclusion and improving tissue perfusion. However, with the paradoxical post‐transfusion decrease in red cell velocity presumably due to induced hyperviscosity from the large transfusion volume, blood flow is still impaired. This decreased velocity may thwart efforts to improve oxygen delivery through transfusion and may, to some extent, promote vasoocclusion instead.

Microvascular abnormalities in the bulbar conjunctiva of contact lens users

Soft contact lenses are commonly used to improve vision acuity or in cosmetic enhancement. We hypothesize that contact lens use can cause inadvertent damage to either the conjunctival microcirculation via direct vasoocclusion when the lens physically interacts with or damages the underlying vessels, or to the bulbar conjunctiva itself when the lens rests unevenly on the surface of the bulbar conjunctiva. Computer-assisted intravital microscopy was utilized to document (via video recording) and objectively quantify (via image analysis) real-time microvascular abnormalities resulting from changes and vessel remodeling in the conjunctival microcirculation in long-term (>2 yrs) contact lens users (n = 102), with non-users serving as control subjects (n = 29). A severity index (SI)--computed as the arithmetic sum of the abnormalities found in the conjunctival microcirculation in each contact lens user--was established for objective comparison with control subjects and critical interpretation. Contact lens user SI was significantly higher than control SI (user = 6.21 ± 1.26; control = 2.31 ± 1.49; p < 0.05), indicative of severe vasculopathy arising from contact lens use. The users also had significantly wider conjunctival vessel diameter (user = 71.25 ± 12.09 μm; control = 52.20 ± 5.10 μm; p < 0.05). Additional abnormalities, including damaged vessels, hemosiderin deposits (from damaged vessels or injury to the surface of the bulbar conjunctiva), vessel sludging, intermittent blood flow, and vessel tortuosity were commonly found in or adjacent to locations where the contact lens physically rested on the underlying conjunctival vessels. These results strongly suggest that microvascular abnormalities and remodeling changes occurred as a result of the inadvertent physical interaction of the lenses with either the underlying conjunctival vessels or the surface of the bulbar conjunctiva in contact lens users.

Correlation of conjunctival microangiopathy with retinopathy in type-2 diabetes mellitus (T2DM) patients

We hypothesized that T2DM vasculopathy can be revealed and quantified in the bulbar conjunctiva prior to its pathologic presentation in the retina. Using computer-assisted intravital microscopy (CAIM), an objective, non-invasive approach can provide a viable complement to retinal fundus photography to possibly screen patients for early signs of real-time, in vivo T2DM vasculopathy. Fundus photography was utilized to determine the retinopathy level (RL) in T2DM patients with non-proliferative diabetic retinopathy (NPDR) and control subjects. CAIM was used to quantify microangiopathy in the bulbar conjunctiva in the same patients, and reported on a severity index (SI). The average RL for the T2DM patients in this study is 19.68 ± 9.91, which differs from control subjects (RL = 10 ± 0.0; p < 0.05). A significant difference in vasculopathy was observed in the conjunctival microcirculation in the same patients (SI = 5.81 ± 1.30) when compared with control subjects (SI = 1.33 ± 1.58; p < 0.05). The results provide evidence that significant vasculopathy had developed in the microcirculation in the bulbar conjunctiva, though diabetic retinopathy had not developed significantly in the same patients - indicative of the presence of a time window for early intervention of T2DM before non-proliferative retinopathy develops, and the real-time availability of the conjunctival microvasculature as an in vivo platform to monitor disease progression.

Effects of GMI-1070, a Pan-Selectin Inhibitor, on Leukocyte Adhesion In Sickle Cell Disease: Results From a Phase 1/2 Study

AbstractAbstract 262Adhesion molecules are critically involved in the pathophysiology of sickle cell disease (SCD). A growing body of evidence from animal models and humans supports the role of selectin-mediated cell adhesion in the pathophysiology of vaso-occlusion. Leukocyte adhesion has been demonstrated to reduce microvascular blood flow in a sickle cell mouse model, and there is evidence to suggest leukocyte adhesion in humans contributes to vaso-occlusion (Stuart et al, Lancet 2004; Turhan et al, PNAS 2002). GMI-1070 is a pan-selectin inhibitor that targets E-, P-, and L-selectins and has previously been shown to restore blood flow and improve survival in a mouse model of vaso-occlusion (Chang et al, Blood 2010). Also, it is potent inhibitor of adhesion of human neutrophils to immobilized E-selectin and ICAM-1 under flow conditions in vitro. As part of a Phase 1/2 study of GMI-1070 in patients with SCD we determined the effects of the drug on biomarkers of adhesion in vivo and ex vivo. Methods: An open-label phase 1/2 study of the safety, PK, and activity of GMI-1070 was performed, enrolling adults with SCD at steady state. Here we are reporting data on GMI-1070 anti-adhesion activity. GMI-1070 was administered in two IV doses given on the same day: 20 mg/kg as a loading dose, followed 10 hours later by 10 mg/kg. Serial WBC count with differential (all 15 subjects), computer-assisted intravital microscopy (CAIM) (4 subjects), and ex vivo activity of GMI-1070 in plasma (4 subjects) were measured. CAIM is a non-invasive technique for quantitative measurement of microvascular blood flow in vessels of the bulbar conjunctiva. In this study it was used to measure RBC velocity before and after dosing (at 30 minutes, 2, 4, 8, and 24 hours) with GMI-1070. Ex vivo evaluation of plasma GMI-1070 activity in a cell adhesion assay was also performed, with samples taken at 0, 8, 24, and 48 hours after first infusion. Results: Adults were enrolled at three centers: 13 with HbSS, 2 with HbSB0thal. All were African-American, and 9 were male. All subjects received both doses of study drug. The t1/2 was 7.7 hours. Mean baseline WBC was 10 K/mm3, and baseline absolute neutrophil count (ANC) was 5.4 K/mm3. The mean WBC was 10.4, 11.6, 10.2, and 8.7 K/mm3 at 8, 24, 48 hours and 7 days, respectively. The ANC mean was 5.5, 7.5, 5.6, and 4.2 K/mm3 at the same time points. ANC % change from baseline was significant at 24 and 48 hours (p=0.001, 0.025 mixed effects model) (Figure). There were no significant changes in absolute monocyte or lymphocyte counts. There was no correlation with any clinical adverse events. In one subject, the WBC rose from 10.4 to a peak of 28, with no clinical symptoms or significant changes in other lab values. CAIM (n=4) evaluating microvascular blood flow in the bulbar conjunctiva (measured in screen pixels/sec), showed mean RBC velocity at baseline was 335 (SD 70) pixels/sec, with mean values at 30 min., 2, 4, and 8 hours of 368 (59), 345 (63), 341 (59), and 338 (83) pixels/sec respectively, and returned to baseline of 317 (82) pixels/sec at 24 h. These differences did not reach statistical significance (mixed effects model). In ex vivo evaluation of neutrophil adhesion to matrix proteins (as quantified by # of bound neutrophils per 50× field) from samples 0, 4, 8, 24, and 48 hours after the first infusion revealed reduction in adhesions at 4 and 8 hours; mean adhered neutrophils were 33 (17), 20 (11), 18 (9), 47 (49), and 42 (15) respectively. However, these differences did not reach statistical significance. In conclusion, GMI-1070 infusion resulted in neutrophilia, a trend towards increased RBC velocity in the vessels of the bulbar conjunctiva immediately after infusion, and reduced leukoctye adhesion in an ex vivo assay despite neutrophilia. All of these findings are consistent with an anti-adhesive effect on leukocyte adhesion in vivo and suggest that the findings in sickle mouse studies can be translated into SCD patients. This study supports further evaluation of GMI-1070 for the treatment of vaso-occlusive episodes in SCD. [Display omitted] Disclosures:Wun:GlycoMimetics: Clinical Trial Sponsorship, Consultancy; Eli Lilly: Clinical Trial Sponsorship, Consultancy. Off Label Use: This drug (GMI-1070) has not been approved for any clinical indication. De Castro:GlycoMimetics: clinical trial sponsorship. Styles:GlycoMimetics: Clinical Trial Sponsorship, Consultancy. Cheung:GlycoMimetics: clinical trial sponsorship. Chase:GlycoMimetics: clinical trial sponsorship. Simon:GlycoMimetics: Research Funding. Magnani:GlycoMimetics: Employment, Equity Ownership. Thackray:GlycoMimetics: Employment, Equity Ownership.

Comparison of real‐time microvascular abnormalities in pediatric and adult sickle cell anemia patients

The conjunctival microcirculation in 14 pediatric and eight adult sickle cell anemia (SCA) patients was studied using computer-assisted intravital microscopy. The bulbar conjunctiva in SCA patients in both age groups exhibited a blanched/avascular appearance characterized by decreased vascularity. SCA patients from both age groups had many of the same abnormal morphometric [vessel diameter, vessel distribution, morphometry (shape), tortuosity, arteriole:venule (A:V) ratio, and hemosiderin deposits] and dynamic [vessel sludging/sludged flow, boxcar blood (trickled) flow, and abnormal flow velocity] abnormalities. A severity index (SI) was computed to quantify the degree of vasculopathy for comparison between groups. The severity of vasculopathy differed significantly between the pediatric and adult patients (SI: 4.2 ± 1.8 vs. 6.6 ± 2.4; P = 0.028), indicative of a lesser degree of overall severity in the pediatric patients. Specific abnormalities that were less prominent in the pediatric patients included abnormal vessel morphometry and tortuosity. Sludged flow, abnormal vessel distribution, abnormal A:V ratio, and boxcar flow appeared in high prevalence in both age groups. The results indicate that SCA microvascular abnormalities develop in childhood and the severity of vasculopathy likely progresses with age. Intervention and effective treatment/management modalities should target pediatric patients to ameliorate, slow down, or prevent progressive microvascular deterioration.

Alzheimer’s disease: More than amyloid

The etiology of Alzheimer's disease (AD) is inconclusive. Treatments targeting amyloid have largely been unsuccessful. There is increasing evidence that vasculopathy may play an important pathogenic role in AD. Longitudinal measurements of whole blood viscosity (WBV) using a computer-assisted hemorheologic protocol and characterization of microvascular abnormalities using computer-assisted intravital microscopy (CAIM) are two objective methods adopted in this laboratory to noninvasively quantify vasculopathy in AD patients. A correlation of increased disease severity with worsened vasculopathy would further bolster a cause and effect relationship. A case report (Case 1) is presented to illustrate the usefulness of following an AD patient with these noninvasive techniques to correlate disease progression with vasculopathy. Patients were selected from a private practice setting who met the Diagnostic and Statistical Manual of Mental Disorders criteria for AD. The Rheolog™, a computer-assisted scanning rheometer, was used to obtain longitudinal measurements of WBV. The microvascular abnormalities in the bulbar conjunctiva were quantified using a severity index (SI, scale 0-15). The patient was observed over a 4 year period from 2005 to 2008. This case study shows a correlation of disease progression in an AD patient with worsened vasculopathy. It illustrates the usefulness of WBV and CAIM as tools to quantify vasculopathy in AD patients and additionally suggests a pathogenetic role vasculopathy may play in concert with the amyloid hypothesis.

Whole blood viscosity and microvascular abnormalities in Alzheimer's Disease

We hypothesized that abnormalities in hemorheologic parameters, including vessel diameter, flow velocity, and whole blood viscosity (WBV), would be present in Alzheimer's Disease (AD) and would correlate with microvascular abnormalities (vasculopathy). Using the Rheolog, we obtained WBV profiles, measured at shear rates of 1-1,000 s-1, for 10 AD subjects and age matched non-AD controls. Vessel diameter, flow velocity, and microvascular abnormalities were quantified using computer-assisted intravital microscopy (CAIM) of the conjunctival microcirculation. A Severity Index (SI), scale 0-15, was computed to reflect degree/severity of vasculopathy. AD subjects compared to controls had significantly higher WBV (3.96+/-0.29 cP vs. 3.34+/-0.05 cP, sheared at 300 s-1; P<0.05) and SI (7.00+/-2.36 vs. 0.30+/-0.70; P<0.05). WBV was correlated (rhos=0.648; P<0.05) with SI in AD subjects. These results strongly suggest the simultaneous involvement of hemorheologic abnormalities and systemic vasculopathy in AD.

Correlation of microvascular abnormalities and endothelial dysfunction in Type-1 Diabetes Mellitus (T1DM): A real-time intravital microscopy study

We hypothesize that real-time in vivo microvascular abnormalities should correlate with biochemical markers of inflammation/endothelial dysfunction in T1DM. Real-time quantification of T1DM and healthy non-diabetic control microcirculation was conducted utilizing computer-assisted intravital microscopy. Selected biochemical markers (high sensitivity C-reactive protein (hsCRP), soluble vascular cell adhesion molecules (sVCAM), soluble intercellular adhesion molecules (sICAM), soluble E-selectin (sE-selectin), nitrotyrosine, superoxide anion (O2-), interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha)) were used for correlation. The severity of microvascular abnormalities, as reflected by the arithmetic severity index (SI), was significantly increased in T1DM vs. controls (5.89 +/- 1.47 vs. 2.34 +/- 1.48; P<0.001). In addition several of the specific microvascular abnormalities (related to flow and morphometry) were significantly more prevalent in the T1DM patients. Finally, the following significant positive correlations existed between the inflammatory/endothelial dysfunction markers and specific microvascular abnormalities: sVCAM and abnormal vessel diameter (P=0.004, OR =1.033, 95% CI for OR =(1.01, 1.056)), superoxide (O2-) release and abnormal vessel distribution (P=0.032, OR =1.798, 95% CI for OR =(1.051, 3.075)), and sE-selectin and abnormal vessel distribution (P=0.036, OR =1.118, 95% CI for OR =(1.007, 1.241)). In view of such significant correlations, we conclude that these specific microvascular abnormalities can serve as unique physiologic markers of endothelial dysfunction to correlate with the biochemical markers of inflammatory/endothelial dysfunction in disease progression and therapeutic efficacy studies.

Hemorheology in Alzheimer's disease: correlation between real‐time microvascular abnormalities and whole blood viscosity

We hypothesized that changes in hemorheologic parameters, including vessel diameter, flow velocity, and whole blood viscosity (WBV), would be present in Alzheimer's disease (AD) and would correlate with microvascular abnormalities (vasculopathy). Using the Rheolog™, we obtained WBV profiles, measured at shear rates of 1‐1,000 s−1, for 10 AD patients and age matched non‐AD controls. Vessel diameter, flow velocity, and vasculopathic changes were quantified using computer‐assisted intravital microscopy (CAIM) of the conjunctival microcirculation. A Severity Index (SI), scale 0–15, was computed to reflect degree of vasculopathy. AD patients had significantly higher WBV (3.96±0.29cp vs. 3.34±0.05cp, sheared at 300s−1; P&lt;0.05) and SI (7.00±2.36 vs. 0.61±0.70; P&lt;0.05) than controls. WBV positively correlated (r=0.68; P&lt;0.05) with SI in AD patients. AD patients with severe dementia had significantly higher WBV (4.23±0.19cp vs. 3.68±0.13cp; P&lt;0.05) and SI (8.60±2.30 vs. 5.4±0.89; P&lt;0.05) than less demented patients. Severity of dementia in AD patients did not necessarily correlate with disease duration, but correlated significantly with WBV and severity of vasculopathy. These results strongly suggest the involvement of hemorheologic changes and peripheral vasculopathy in AD.

Evidence of Increased Inflammation and Microcirculatory Abnormalities in Patients With Type 1 Diabetes and Their Role in Microvascular Complications

Type 1 diabetes is associated with increased microvascular complications and inflammation. The monocyte-macrophage is a pivotal cell in atherogenesis. There are scanty data on noninvasive measures of microvascular abnormalities and inflammation in type 1 diabetic subjects with microvascular complications. Thus, we examined systemic and cellular biomarkers of inflammation in type 1 diabetic patients with microvascular complications (T1DM-MV patients) and type 1 diabetic patients without microvascular complications (T1DM patients) compared with matched control subjects and determined the microcirculatory abnormalities in the T1DM and T1DM-MV patients using computer-assisted intravital microscopy (CAIM). Fasting blood, 24-h urine, and CAIM measurements were obtained from the T1DM and T1DM-MV patients and matched control subjects. C-reactive protein, E-selectin, nitrotyrosine, monocyte superoxide, and cytokines were elevated in the T1DM and T1DM-MV patients compared with control subjects (P < 0.01). Severity index, as assessed by CAIM, was significantly increased in the T1DM and T1DM-MV patients compared with the control subjects (P < 0.001). There was a significant increase in C-reactive protein, nitrotyrosine, vascular cell adhesion molecule and monocyte superoxide anion release, and interleukin-1 release in T1DM-MV compared with T1DM patients (P < 0.05). T1DM-MV patients had significantly increased CAIM severity index and microalbumin-to-creatinine ratio compared with T1DM patients (P < 0.05). Furthermore, pp38MAPK, pp65, and pERK activity were significantly increased in monocytes from the T1DM and T1DM-MV patients compared with those from the controls subjects, and pp38MAPK and pp65 activity were significantly increased in the T1DM-MV compared with the T1DM patients (P < 0.01). T1DM-MV patients have increased inflammation compared with T1DM patients. CAIM provides an effective biomarker of microvascular complications, since it is significantly elevated in T1DM-MV compared with T1DM patients and can be monitored following therapies targeted at improving inflammation and/or microvascular complications of type 1 diabetes.

A novel approach to the study of human microcirculation: reactivity to locally applied angiotensin II in the conjunctival microvascular bed

To develop a state-of-the-art, computer-assisted intravital microscopy protocol to evaluate directly the effects of topically applied drugs on conjunctival arteriolar and venular diameters. Fifty-one normotensive volunteers were studied. Video-recordings of the bulbar conjunctival microcirculation were made before and following eye drops containing angiotensin II (AngII) (0.001% w/w, or 0.01%) or phenylephrine (0.25%). The computer-assisted analyses of arteriolar and venular diameters were performed off-line. In different protocols the microvascular reactivity to the different eye drops were compared. AngII (0.01%) eye drops, but not AngII (0.001%), induced significant constriction in both arterioles (median, 19%) and venules (13%). Phenylephrine eye drops (pharmacological control) induced similar arteriolar (18%) and venular (12%) constrictions. Repeated AngII challenges with a 30-min interval revealed reproducible vasoconstriction responses (median arteriolar constriction, 11 and 17%, respectively; NS). The vasoconstriction responses following AngII challenges on two consecutive days revealed reproducible responses (median arteriolar constriction, 13 and 11%, respectively; NS). The present results demonstrate that the proposed model for noninvasive intravital video-microscopy of the conjunctival microcirculation is sensitive for measuring direct arteriolar and venular reactivity following topically applied drugs. We consider this model a valuable tool for sophisticated research on in-vivo microvascular reactivity in humans.

Microvascular abnormalities and whole blood viscosity in Alzheimer's disease

Microvascular abnormalities (MA) and whole blood viscosity (WBV) in Alzheimer's disease (AD) patients have been studied for direct correlation. Computer-assisted intravital microscopy (CAIM) was used to objectively quantify MA in the conjunctival microcirculation in AD patients (n=12), using healthy volunteers as control subjects (n=10). Fifteen recognizable MA existed in various vascular diseases, and 10 were commonly found in AD patients, though not all in the same patient. In addition, MA were not found in control subjects. A severity index (SI) -- the arithmetic sum of MA in each patient quantified via CAIM -- was computed to give an objective and quantitative score to correlate with medical history, disease severity, WBV and shear rates. AD SI (7.2±2.8) differed significantly (P<0.05) from control SI (0.6±0.7) and correlated significantly with disease severity. The RheologTM, a computer-assisted scanning rheometer, was used to generate a WBV profile over a 1 to 1,000s−1 range of shear rates. AD WBV (at various shear rates) differed significantly from control values. For example, AD WBV at a shear rate of 300s−1 (3.81±0.37cp) differed significantly (P<0.05) from control subjects (3.34±0.05cp). AD WBV correlated with SI and disease severity, but not with duration of the disease since diagnosis. This finding is intriguing as it indicates that SI and WBV, when combined, may represent a unique availability of correlated quantitative markers with which to predict disease progression or outcome. Funded by a discretion gift from Rheologics, Inc. and a UC Davis Faculty Research Award.

Transgenic Sickle Mice Are Markedly Sensitive to Renal Ischemia-Reperfusion Injury

Ischemic injury is invoked as a mechanism contributing to end-organ damage and other complications of sickle cell disease (SCD). However, the intrinsic sensitivity of tissues in SCD to ischemic insults has never been addressed. We examined the effect of renal ischemia in a transgenic mouse expressing human sickle hemoglobin. Twenty-four hours after bilateral, total renal artery occlusion for 15 minutes, transgenic sickle mice exhibited worse renal function and more marked histological injury. With bilateral renal ischemia of greater duration (22.5 minutes), and after 6 hours, transgenic sickle mice exhibited massive vascular congestion, sickling of red blood cells, more marked histological injury in the kidney, and more prominent congestion in the capillary beds in the lungs and heart. Additionally, serum amyloid P-component, the murine homologue of C-reactive protein, was markedly increased in transgenic sickle mice as compared to wild-type mice. Twenty-four hours after bilateral renal ischemia for 22.5 minutes, transgenic sickle mice exhibited 28% mortality, with no mortality observed in any other group. With bilateral renal ischemia of short or long duration, renal expression of caspase-3 was most prominent in transgenic sickle mice subjected to ischemia. Thus, renal ischemia in this murine model induces more severe renal injury and extrarenal complications. We conclude that tissues in SCD exhibit heightened vascular congestion and sensitivity to ischemia and that clinically apparent or silent episodes of ischemia may contribute to the complications of SCD.