An integrin binding-defective mutant of insulin-like growth factor-1 (R36E/R37E IGF1) acts as a dominant-negative antagonist of the IGF1 receptor (IGF1R) and suppresses tumorigenesis but still binds to IGF1R.

Insulin-like growth factor-1 (IGF1) is a major therapeutic target for cancer. We recently reported that IGF1 directly binds to integrins (αvß3 and α6ß4) and induces ternary complex formation (integrin-IGF1-IGF1 receptor (IGF1R)) and that the integrin binding-defective mutant of IGF1 (R36E/R37E) is defective in signaling and ternary complex formation. These findings predict that R36E/R37E competes with WT IGF1 for binding to IGF1R and inhibits IGF signaling. Here, we described that excess R36E/R37E suppressed cell viability increased by WT IGF1 in vitro in non-transformed cells. We studied the effect of R36E/R37E on viability and tumorigenesis in cancer cell lines. We did not detect an effect of WT IGF1 or R36E/R37E in cancer cells under anchorage-dependent conditions. However, under anchorage-independent conditions, WT IGF1 enhanced cell viability and induced signals, whereas R36E/R37E did not. Notably, excess R36E/R37E suppressed cell viability and signaling induced by WT IGF1 under anchorage-independent conditions. Using cancer cells stably expressing WT IGF1 or R36E/R37E, we determined that R36E/R37E suppressed tumorigenesis in vivo, whereas WT IGF1 markedly enhanced it. R36E/R37E suppressed the binding of WT IGF1 to the cell surface and the subsequent ternary complex formation induced by WT IGF1. R36E/R37E suppressed activation of IGF1R by insulin. WT IGF1, but not R36E/R37E, induced ternary complex formation with the IGF1R/insulin receptor hybrid. These findings suggest that 1) IGF1 induces signals under anchorage-independent conditions and that 2) R36E/R37E acts as a dominant-negative inhibitor of IGF1R (IGF1 decoy). Our results are consistent with a model in which ternary complex formation is critical for IGF signaling.

A potent oral P-selectin blocking agent improves microcirculatory blood flow and a marker of endothelial cell injury in patients with sickle cell disease.

Abnormal blood flow accounts for most of the clinical morbidity of sickle cell disease (SCD) [1,2]. Most notably, occlusion of flow in the microvasculature causes the acute pain crises [3] that are the commonest cause for patients with SCD to seek medical attention [4] and major determinants of their quality of life [5]. Based on evidence that endothelial P-selectin is central to the abnormal blood flow in SCD we provide results from four of our studies that are germane to microvascular blood flow in SCD. A proof-of-principle study established that doses of heparin lower than what are used for anticoagulation but sufficient to block P-selectin improved microvascular blood flow inpatients with SCD. An in vitro study showed that Pentosan Polysulfate Sodium (PPS) had greater P-selectin blocking activity than heparin. A Phase I clinical study demonstrated that a single oral dose of PPS increased microvascular blood flow in patients with SCD. A Phase II clinical study that was not completed documented that daily oral doses of PPS administered for 8 weeks lowered plasma levels of sVCAM-1 and tended to improve microvascular blood flow in patients with SCD. These data support the concept that P-selectin on the microvascular endothelium is critical to both acute vascular occlusion and chronically impaired microvascular blood flow in SCD. They also demonstrate that oral PPS is beneficial to microvascular sickle cell blood flow and has potential as an efficacious agent for long-term prophylactic therapy of SCD.

Exchange transfusion therapy and its effects on real-time microcirculation in pediatric sickle cell anemia patients: an intravital microscopy study.

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.

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.

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.

CONTEXT: 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. OBJECTIVE: 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. DESIGN: 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. CONCLUSION: 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.

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.

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.

Effects of pegylated hamster red blood cells on microcirculation.

The objective of this study was to examine the effects of polyethylene glycol (PEG) treated red blood cells (RBCs) on the microcirculation in a hamster back skin window chamber model. Donor hamster RBCs were PEGylated through an incubation with an activated PEG solution, washed, resuspended, and infused through a 10% volume top loading procedure into the carotid artery in an awake Syrian Golden hamster. Eight hamster groups were treated with activated PEG different sizes and concentrations: 0.05 mM-5 kDa PEG, 0.5 mM-5 kDa PEG, 1.1 mM-5 kDa PEG, 2.2 mM-5 kDa PEG, 22 mM-5 kDa PEG, 0.05 mM-20 kDa PEG, 0.5 mM-20 kDa PEG, and 5 mM-20 kDa PEG. Non-treated RBCs were used as control. The microvascular bed under observation was videotaped 30 min before the infusion and followed for 30 min post infusion. The diameter of individual blood vessels and blood flow velocities in selected vessels was measured. Hematocrit and hemoglobin concentration were recorded before infusion and at the end of experiment. Tissue pO(2) was also monitored. Results showed the hamsters tolerated the PEGylated RBCs without apparent ill effects. No significant changes were recorded for the hematocrit, the hemoglobin concentration, the blood vessel diameters, blood flow velocities, and the interstitial partial oxygen pressure (pO(2)) before, during, and after the injections of PEG-RBCs (P > 0.05). Unlike most hemoglobin-based oxygen carrying compounds, which can cause vasoconstriction, the PEGylated RBCs did not produce any measurable vasoactivity. Together with the absence of rouleaux formation and the fact that PEG molecules can mask the surface antigens on RBCs, PEGylation appeared promising as a circulation enhancement treatment.

Effects of low-volume hemoglobin glutamer-200 versus normal saline and arginine vasopressin resuscitation on systemic and skeletal muscle blood flow and oxygenation in a canine hemorrhagic shock model.

OBJECTIVE: To test the hypothesis that low-volume resuscitation with hemoglobin glutamer-200 improves hemodynamic function and tissue oxygenation, whereas arginine vasopressin resuscitation improves blood pressures more than low-volume saline or hemoglobin glutamer infusion but compromises systemic and muscle blood flow and oxygenation. DESIGN: Randomized laboratory investigation. SETTING: University research facility. SUBJECTS: Nineteen dogs. INTERVENTIONS: Dogs were instrumented to determine heart rate; arterial, central venous, pulmonary arterial, and pulmonary arterial occlusion pressures; cardiac output; and quadriceps muscle blood flow and oxygen tension (PMo2). Total and plasma hemoglobin, oxygen content, lactate, pH, standard base excess, and arginine vasopressin levels were determined, and systemic oxygen delivery (Do2I) and extraction ratio were calculated. Measurements were made before and 30 mins following hemorrhage. Dogs were resuscitated over 60 mins with saline (8.5 mL/kg), arginine vasopressin (0.4 IU/kg bolus plus 0.08 IU x kg x min), or 1:1 diluted hemoglobin glutamer-200. Recordings were then repeated. Subsequently, animals received 30 mL/kg shed blood (60 mL x kg x hr), and recordings were repeated immediately and 1 hr later. MEASUREMENTS AND MAIN RESULTS: Hemorrhage ( approximately 52 mL/kg) caused characteristic changes in hemodynamic, hematologic, systemic PMo2, and acid-base variables. Saline resuscitation increased both Do2I and muscle perfusion by 42% and 51%, while arginine vasopressin treatment reduced heart rate by 31% and increased mean arterial pressure by 22% but not cardiac output, Do2I, or muscle blood flow, resulting in a further decrease of PMo2 by 68% and worse metabolic acidosis. Hemoglobin glutamer-200 infusion caused systemic and pulmonary vasoconstriction, however, without deterioration of cardiac output, Do2I, muscle blood flow, or PMo2 despite lack of oxygen content increase. Blood transfusion restored most variables. CONCLUSIONS: Low-volume crystalloid or hemoglobin glutamer-200 resuscitation posthemorrhage may improve (but not restore) macro- and microvascular functions and tissue oxygenation, while arginine vasopressin infusion may only improve blood pressures and result in lower overall systemic perfusion compared with low-volume saline or hemoglobin glutamer-200 treatment and worsening of anaerobic conditions in skeletal muscle.

Comparison of treatment modalities for hemorrhagic shock.

Allogeneic blood resuscitation is the treatment of choice for hemorrhagic shock. When blood is unavailable, plasma expanders, including crystalloids, colloids, and blood substitutes, may be used. Another treatment modality is vasopressin, a vasoconstrictor administered to redistribute blood flow, increase venous return, and maintain adequate cardiac output. While much information exists on systemic function and oxygenation characteristics following treatment with these resuscitants, data on their effects on the microcirculation and correlation of real-time microvascular changes with changes in systemic function and oxygenation in the same animal are lacking. In this study, real-time microvascular changes during hemorrhagic shock treatment were correlated with systemic function and oxygenation changes in a canine hemorrhagic shock model (50-55% total blood loss with a MAP of 45-50 mmHg as a clinical criterion). Following splenectomy and hemorrhage, the dogs were assigned to five resuscitation groups: autologous/shed blood, hemoglobin-based oxygen carrier/Oxyglobin, crystalloid/saline, colloid/Hespan (6% hetastarch), and vasopressin. Systemic function and oxygenation changes were continuously monitored and periodically measured (during various phases of the study) using standard operating room protocols. Computer-assisted intravital video-microscopy was used to objectively analyze and quantify real-time microvascular changes (diameter, red-cell velocity) in the conjunctival microcirculation. Measurements were made during pre-hemorrhagic (baseline), post-hemorrhagic (pre-resuscitation), and post-resuscitation phases of the study. Pre-hemorrhagic microvascular variables were similar in all dogs (venular diameter = 42+/-4 microm, red-cell velocity = 0.55+/-0.5 mm/sec). All dogs showed significant (P < 0.05) post-hemorrhagic microvascular changes: approximately 20% decrease in venular diameter and approximately 30% increase in red-cell velocity, indicative of sympathetic effects arising from substantial blood loss. Microvascular changes correlated with post-hemorrhagic systemic function and oxygenation changes. All resuscitation modalities except vasopressin restored microvascular and systemic function changes close to pre-hemorrhagic values. However, only autologous blood restored oxygenation changes to pre-hemorrhagic levels. Vasopressin treatment resulted in further decreases in venular diameter (approximately 50%) as well as red-cell velocity (approximately 70%) without improving cardiac output. Our results suggested that volume replenishment - not oxygen-carrying capability - played an important role in pre-hospital/en route treatment for hemorrhagic shock. Vasopressin treatment resulted in inadvertent detrimental outcome without the intended benefit.

Effects of poloxamer 188 treatment on sickle cell vaso-occlusive crisis: computer-assisted intravital microscopy study.

Nine patients with sickle cell disease (SCD) who were hospitalized at UC Davis Medical Center for vaso-occlusive crisis (VOC) were studied as part of a randomized double-blind phase III clinical trial to investigate the real-time effects of poloxamer 188 on VOC. All patients showed significant microvascular changes from normal (steady-state) values during VOC (diminished venular diameter and red cell velocity). The patients were randomly assigned to be treated with poloxamer 188 or placebo. Poloxamer 188 (n = 4) but not placebo (n = 5) significantly reversed these microvascular changes approximately 2 hours postinitiation of loading infusion (100 mg/kg in 1 hour). Further significant improvement induced by poloxamer 188 but not placebo was observed > or = 7 hours postinfusion, resulting in a significant reversal of the microvascular changes to steady-state values.

The contribution of endothelial cell P-selectin to the microvascular flow of mouse sickle erythrocytes in vivo.

Microvascular occlusion in sickle cell disease can be initiated by adhesion of sickle red blood cells (RBCs) to the endothelium. Our objective in this study was to verify the relevance in vivo of our discovery that sickle RBCs adhere abnormally to endothelial P-selectin in vitro. We used computer-assisted intravital microscopy to characterize RBC flow velocity (V(RBC)) in mice. We found faster V(RBC) of sickle RBCs in P-selectin knock-out and control mice than in sickle cell mice, which have increased endothelial cell P-selectin expression. Agonist peptide for murine protease-activated receptor-1 (PAR-1), which selectively activates mouse endothelial cells but not platelets, was used to assess the effects of endothelial cell P-selectin on microvascular flow. Suffusion of venules with this agonist stopped flow promptly in normal and sickle mice but not in P-selectin knock-out mice or in control mice pretreated with anti-P-selectin monoclonal antibody or unfractionated heparin (UFH). Agonist-induced slowing of flow was reversed rapidly by suffusion with UFH, provided flow had not already stopped. We conclude that endothelial cell P-selectin contributes to the microcirculatory abnormalities in sickle cell disease and that blocking P-selectin may be useful for preventing painful vasoocclusion in sickle cell disease.

Blood substitute resuscitation as a treatment modality for moderate hypovolemia.

Blood substitute resuscitation as a treatment modality for moderate hypovolemia (approximately 40% blood loss) in a canine model has been evaluated using Oxyglobin (Biopure Hemoglobin Glutamer-200/ Bovine; a hemoglobin-based oxygen-carrier) and Hespan (6% hetastarch; a nonoxygen-carrier) as resuscitants. Autologous (shed) blood served as control. Nine dogs were studied--after splenectomy, each dog was hemorrhaged (32-36 mL/kg; MAP = approximately 50 mmHg) and randomly assigned to the three resuscitation groups. Microvascular, systemic function and oxygenation characteristics were monitored and/or measured simultaneously in prehemorrhagic (baseline), posthemorrhagic and postresuscitation phases for correlation-real-time microvascular changes in the bulbar conjunctiva were noninvasively measured via computer-assisted intravital microscopy and systemic function and oxygenation changes were monitored and/or measured via instrumentation and devices incorporated into our bioengineering station in an operating room setting. Blood chemistry was also studied for relevant measurements. Prehemorrhagic microvascular characteristics were similar in all animals (venular diameter = 41 +/- 12 microm, A:V ratio = approximately 1:2, red-cell velocity = 0.5 +/- 0.3 mm/s). All animals also showed similar prehemorrhagic systemic function and oxygenation measurements comparable to a previous study and were consistent with normal measurements in dogs. At the completion of hemorrhaging to achieve moderate hypovolemia (approximately 40% blood loss with MAP at approximately 50 mmHg), all nine animals showed similar significant (P < 0.01) posthemorrhagic microvascular changes, including approximately 17% decrease in diameter (34 +/- 7 microm), A:V ratio = variable, and approximately 80% increase in velocity (0.9 +/- 0.5 mm/s). All animals also showed similar significant (P < 0.01) posthemorrhagic systemic function and oxygenation changes, with decreases in Hct, aHb(total), MPAP, MAP, SAP, DAP, CO, SVI, CaO2, and CvO2 and increases in HR and lactic acidosis. Shed blood (control) resuscitation restored posthemorrhagic microvascular changes close to prehemorrhagic values (diameter = 39 +/- 6 microm, A:V ratio = approximately 1:2, velocity = 0.6 +/- 0.4 mm/s). Oxyglobin and Hespan restored microvascular changes in similar manner close to prehemorrhagic values (Oxyglobin: diameter = 38 +/- 3 microm, A:V ratio = approximately 1:2, velocity = 0.6 +/- 0.4 mm/s; Hespan: diameter = 38 +/- 7 microm, A:V ratio = 1:2, velocity = 0.5 +/- 0.4 mm/s). After resuscitation, shed blood (control) restored all systemic function and oxygenation changes close to prehemorrhagic values. However, both Oxyglobin and Hespan resuscitation restored systemic function changes, but not oxygenation changes, to prehemorrhagic values. This was an interesting finding because of the different oxygen-carrying capability of Oxyglobin (oxygen-carrying) and Hespan (nonoxygen-carrying). The result suggests that either volume replenishment alone (and not oxygen-carrying capability) is needed to treat moderate hypovolemia or oxygenation measurements obtained by standard methods (oximetry, blood chemistry) may not reflect tissue oxygenation levels.

Microvascular abnormalities in sickle cell disease: a computer-assisted intravital microscopy study.

The conjunctival microcirculation of 18 homozygous sickle cell disease (SCD) patients during steady-state, painful crisis, and postcrisis conditions was recorded on high-resolution videotapes using intravital microscopy. Selected videotape sequences were subsequently coded, frame-captured, studied, and blindly analyzed using computer-assisted image analysis protocols. At steady-state (baseline), all SCD patients exhibited some of the following morphometric abnormalities: abnormal vessel diameter, comma signs, blood sludging, boxcar blood flow phenomenon, distended vessels, damaged vessels, hemosiderin deposits, vessel tortuosity, and microaneurysms. There was a decrease in vascularity (diminished presence of conjunctival vessels) in SCD patients compared with non-SCD controls, giving the bulbar conjunctiva a "blanched" avascular appearance in most but not all SCD patients during steady-state. Averaged steady-state red cell velocity in SCD patients was slower than in non-SCD controls. During painful crisis, a further decrease in vascularity (caused by flow stoppage in small vessels) and a 36.7% +/- 5.2% decrease in large vessel (mostly venular) diameter resulted. In addition, the conjunctival red cell velocities either slowed significantly (6.6% +/- 13.1%; P <.01) or were reduced to a trickle (unmeasurable) during crisis. The microvascular changes observed during crisis were transient and reverted to steady-state baseline after resolution of crisis. When combined, intravital microscopy and computer-assisted image analysis (computer-assisted intravital microscopy) represent the availability of a noninvasive tool to quantify microvascular abnormalities in vascular diseases, including sickle cell disease. The ability to identify and relocate the same conjunctival vessels for longitudinal studies uniquely underscores the applicability of this quantitative real-time technology.

Microvascular abnormalities in pediatric diabetic patients.

Microvascular abnormalities are associated with and causative of the development of end-stage organ complications in adult diabetic patients. Whether the same microvascular abnormalities are present in pediatric patients is not known and has not been studied because of a lack of real-time technology, methodology to study young patients, and availability of an appropriate noninvasive site for in vivo studies. We hypothesized that microvascular abnormalities should be present in pediatric patients despite their young age and the relatively short durations of the disease. In this study, computer-assisted intravital microscopy (CAIM) was adapted to blindly quantify microvascular abnormalities in 12 pediatric type 1 diabetic mellitus (T1DM) patients (ages = 6-16 years; mean +/- SD = 11.42 +/- 3.42; duration since diagnosis = 2-14 years; mean +/- SD = 6.75 +/- 3.79) in vivo, using the microcirculation of the bulbar conjunctiva as a noninvasive site. Microvascular abnormalities, commonly found in adult patients, existed in the conjunctival microcirculation of all pediatric T1DM patients in varying degrees despite their relatively young age. A severity index (SI) was developed to reflect the cumulative severity of the microvascular abnormalities and was computed as the summation of all microvascular abnormalities found in each patient. SI for the 12 T1DM patients (mean +/- SD = 7.42 +/- 1.88; median = 8; mode = 9) differed significantly from that for the nondiabetic controls (mean +/- SD = 0.67 +/- 0.78; median = 0.5; mode = 0; P < 0.0001). In addition, SI correlated with hemoglobin A1c levels (mean +/- SD = 9.18 +/- 1.57) of T1DM patients but did not correlate with the duration of disease since diagnosis of the same patients. This observation raises the possibility that diabetic pathogenesis may precede the onset of overt disease or clinical diagnosis. This study confirms that CAIM may represent the availability of a useful real-time technology to study conjunctival microvascular abnormalities in vascular diseases in juvenile as well as adult patients.