microvascular abnormalities in pediatric diabetic patients

Microvascular Abnormalities in PediatricDiabetic Patients

Anthony T. W. Cheung,*,† Amber R. Price,* Patricia L. Duong,*Sahana Ramanujam,* Jana Gut,* Edward C. Larkin,*Peter C. Y. Chen,‡ and Darrell M. Wilson†*Department of Medical Pathology, University of California, Davis School of Medicine, Davis, California 95616;‡Department of Bioengineering, University of California, San Diego, La Jolla, California 92122; and†Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305

Microvascular abnormalities are associated with andcausative of the development of end-stage organ compli-cations in adult diabetic patients. Whether the same mi-crovascular abnormalities are present in pediatric pa-tients is not known and has not been studied because of alack of real-time technology, methodology to study youngpatients, and availability of an appropriate noninvasivesite for in vivo studies. We hypothesized that microvas-cular abnormalities should be present in pediatric pa-tients despite their young age and the relatively shortdurations of the disease. In this study, computer-assistedintravital microscopy (CAIM) was adapted to blindlyquantify microvascular abnormalities in 12 pediatrictype 1 diabetic mellitus (T1DM) patients (ages � 6–16years; mean � SD � 11.42 � 3.42; duration since diag-nosis � 2–14 years; mean � SD � 6.75 � 3.79) in vivo,using the microcirculation of the bulbar conjunctiva as anoninvasive site. Microvascular abnormalities, com-monly found in adult patients, existed in the conjunctivalmicrocirculation of all pediatric T1DM patients in vary-ing degrees despite their relatively young age. A severityindex (SI) was developed to reflect the cumulative sever-
ity of the microvascular abnormalities and was computedas the summation of all microvascular abnormalitiesfound in each patient. SI for the 12 T1DM patients
252

(mean � SD � 7.42 � 1.88; median � 8; mode � 9)differed significantly from that for the nondiabetic con-trols (mean � SD � 0.67 � 0.78; median � 0.5; mode �

0; P < 0.0001). In addition, SI correlated with hemoglo-bin A1c levels (mean � SD � 9.18 � 1.57) of T1DMpatients but did not correlate with the duration of diseasesince diagnosis of the same patients. This observationraises the possibility that diabetic pathogenesis may pre-cede the onset of overt disease or clinical diagnosis. Thisstudy confirms that CAIM may represent the availabilityof a useful real-time technology to study conjunctivalmicrovascular abnormalities in vascular diseases in juve-nile as well as adult patients. © 2002 Elsevier Science (USA)

Key Words: pediatric T1DM; in vivo microvascular ab-normalities; computer-assisted intravital microscopy; se-verity index.

INTRODUCTION

In all systemic vascular disorders, the microcircula-tion normally shows substantial morphometric, struc-

Received May 14, 2001

Microvascular Research 63, 252–258 (2002)doi:10.1006/mvre.2001.2386, available online at http://www.idealibr
on ary.com
tural, and dynamic alterations and adaptations, de-pending on the disease state. Such changes eventuallydisrupt or compromise tissue integrity, leading to or-

0026-2862/02 $35.00© 2002 Elsevier Science (USA)

All rights reserved.

gan and tissue damage or complications. One canconsider vascular disease as a departure from thenormal state in which the microvascular networks inthe body compensate by undergoing varying degreesof remodeling as a response to most transient distur-bances and to maintain local homeostasis (Zweifach,1994, 1995). The body can normally adjust to transientdisturbances. However, when these disturbances be-come chronic (as in diabetes mellitus, sickle cell dis-ease, and hypertension), active adjustments will grad-ually give way to adaptations of blood vesselgeometry and structure (microvascular abnormali-ties/microangiopathy). Existing data on adult diabeticpatients suggest that the presence of microvascularabnormalities may reflect disease severity. Further-more, it is likely that manifestations in the microvas-cular networks may represent the characteristic signsof disease adaptations and may parallel the emergenceof recognizable clinical sequelae (Zweifach, 1994,1995).

Although much research has been conducted inadult diabetic patients, there is a paucity of informa-tion on microvascular complications in pediatric pa-tients. Because of the lack of real-time technology,methodology to study toddlers and juveniles, andavailability of a relevant site(s) for noninvasive studieson pathogenesis of diabetic microangiopathy, in vivoresearch on pediatric microvascular abnormalities hasrarely been performed. A longitudinal study has beeninitiated to study pediatric diabetic patients and sib-lings using computer-assisted intravital microscopy(CAIM), a real-time quantitative technology, whichwas previously described in a report on the reversal ofdiabetic microangiopathy in vivo in pancreas–kidneytransplanted patients (Cheung et al., 1999). The effi-cacy and applicability of this quantitative real-timetechnology have been tested and utilized successfullyin studying type 2 diabetes mellitus (T2DM) patientsin vivo in another study (Cheung et al., 2001). Thissame technology has now been adapted for use intoddlers and juvenile patients.

In this study, we hypothesized that microvascularabnormalities should be present in the conjunctivalmicrocirculation of pediatric diabetic patients despitetheir young age and the relatively short durations ofthe disease since diagnosis.

METHODS

Experimental Subjects

Pediatric type 1 diabetes mellitus (T1DM) patients(n � 12; ages � 6–16 years; mean � SD � 11.42 �3.42; duration since diagnosis � 2–14 years; mean �SD � 6.75 � 3.79) were recruited from the StanfordUniversity Lucille Packard Children’s Hospital Diabe-tes Clinic for the study. Prior to the study, each of thepatients was given a complete physical examinationand was determined to be free of health problems notarising directly from T1DM. Healthy nondiabetic sub-jects were used as controls and were confirmed tohave no personal or family history of vascular dis-eases. Informed consent was obtained from the pa-tients or controls over 12 years of age and also fromthe parents or guardians prior to the study. This studywas approved by the Human Subjects Use Commit-tees (IRB) in Stanford University and University ofCalifornia, Davis and was in accordance with the Dec-laration of Helsinki.

Computer-Assisted Intravital Microscopy

The conjunctival microcirculation was studied in allT1DM patients and normal control subjects usingCAIM (Cheung et al., 1999, 2001). The T1DM video-tape procedure was conducted at Stanford Universityby the principal investigator/first author. The video-tape procedure for the control subjects was conductedat UC Davis Medical Center. The principal investiga-tor was blinded to the medical history of all patientsand control subjects. A venous blood sample of eachpatient was obtained for hemoglobin A1c (HbA1c)measurement (Stanford Clinical Laboratory) on thesame day of the study.

The CAIM procedure was described in detail inprevious reports (Chen et al., 1987; Cheung et al., 1996,1997, 1999, 2001). Briefly, the microcirculation of thebulbar conjunctiva was videotaped in each experi-mental subject using a charge-coupled device (CCD)video camera (COHU Model CCD-6415-3000). A fiber-optics light source (Fiber-Lite Model 3100) with aKodak No. 58 Wratten (anti-red) filter was focused on

253Microangiopathy in Pediatric T1DM

© 2002 Elsevier Science (USA)All rights reserved.

the peri-limbal vessels of the bulbar conjunctiva forepi-illumination. Other regions (fornix or corner) ofthe bulbar conjunctiva were not studied because ofinherent microvascular variability. Each experimentalsubject (patient or control) was seated and asked torelax for at least 5 min. During this period, he/she wascautioned not to touch or rub the eye during relax-ation. If there was any irritation or discomfort, twodrops of nonmedicated ophthalmic saline solutionwere applied and excessive saline was blotted off bytissue at the corner of the eye. The subject again re-laxed before being videotaped, with his/her head rest-ing on a chin–forehead restraint and elbows restingsteadily on the bench where the restraint was securelymounted. The height of the CAIM system was ad-justed to align horizontally with the peri-limbal regionof the eye (left eye was normally used) at an anglewhich provided the flattest surface for focusing. Oncein focus, the conjunctival vessels appeared as sharpblack lines and tubes on screen. A 15-min videotapesequence was normally made of each experimentalsubject and constant on-screen refocusing was con-ducted to ensure sharp image display in at least fivedifferent fields. When in focus, the front optical ele-ment of CAIM was �6 cm from the peri-limbal region(8.53 mm2; 4.5� optical magnification) without havingto touch or displace the eyelid. The videotapes on theconjunctival microcirculation in all experimental sub-jects were made at Stanford University. In order tomaintain objectivity during data analysis, all videosequences were coded and sent to the Image AnalysisLaboratory at UC Davis Medical Center for blindedanalysis.

All coded video sequences were studied in theirentirety to identify morphometric microvascular ab-normalities in the conjunctival microcirculation, withthe identity of the patients and their medical recordsblinded to the investigators. Normally, five or morevideo sequences (with at least one video sequencefrom each of the five different fields videotaped) fromeach patient and control subject were selected. A well-resolved video frame from each video sequence wascaptured for detailed analysis. Morphometric abnor-malities, including abnormal vessel morphometry(beaded vessel), vessel tortuosity, box car blood flowphenomenon, damaged vessel, hemosiderin deposit,

and distended vessel, were separately scored by threeinvestigators. If any morphometric abnormality wasfound in three or more video frames, the abnormalitywould be confirmed as present in that patient. Alldiscrepancies, though rare, were discussed and recon-ciled by the investigators.

After the identification of all morphometric abnor-malities, the same five or more coded video frameswere further analyzed and objectively quantified us-ing in-house developed imaging software (VASCANand VASVEL) (Chen et al., 1987; Cheung et al., 1996,1997, 1999, 2001). VASCAN was used to quantify ad-ditional microvascular characteristics of each patient,including averaged vessel diameters, arteriole:venule(A:V) ratio, ischemic areas, and vessel density distri-bution. Each captured video frame (8.53 mm2) wasalso analyzed for individual vessel diameters and ves-sel tortuosity using VASVEL. In addition, the videosequences were analyzed in consecutive frames (eightsuccessive frames) to determine blood flow velocityusing VASVEL. The computer-generated measure-ments of each T1DM patient were compared with thecontrol measurements for statistical significance (P �

0.05). If significance was confirmed, the presence ofthe abnormality was confirmed objectively to exist inthe patient.

Fifteen recognizable microvascular abnormalitieswere found in the conjuctival microcirculation in pre-vious diabetic studies (Cheung et al., 2001). These in-clude abnormal vessel diameter, thickened vesselwall, abnormal vessel morphometry (beaded vessel),vessel tortuosity (meandering vessel), blood sludging,distended vessel, damaged vessel, hemosiderian de-posit, microaneurysm (micropool), abnormal vesseldistribution, abnormal A:V ratio, avascularity (isch-emic area), comma sign (blocked vessel), box car bloodflow phenomenon, and abnormal blood flow velocity.However, not all 15 abnormalities were present in anysingle patient in this study; some of the 15 abnormal-ities were present in each patient and the sum total ofthe abnormalities should reflect disease severity in thepatient. In order to quantify the total number of ab-normalities present in a patient and for ease of corre-lation, a severity index (SI) has been established basedon the following computation: the presence of eachconfirmed microvascular abnormality in a patient

254 Cheung et al.


would be given a score of “1,” with a highest possible(theoretical) SI of “15” and a lowest possible SI of “0.”

The results obtained in the blinded analysis at UCDavis Medical Center were sent to Stanford Universitywhere all medical records were kept. The medicalhistory of each patient (including disease severity,disease progression and management, HbA1c levels,and duration of disease since diagnosis) was matchedwith the microvascular results and SI of the samepatient for data interpretation and correlation.

Statistics

Results were averaged and are reported as means �SD. For statistical analysis, analysis of variance(ANOVA) was used and a 0.05 significance level wasadopted in this study.

RESULTS

Using intravital microscopy, capillaries, arterioles,and venules appeared as black lines or tubes on awhite background (Figs. 1A–1D). Blood flow could beseen in most arterioles and small venules, but was lessvisible in the larger venules. Each patient was ob-served to have some of the 15 microvascular abnor-malities identified in T2DM patients in a previousstudy (Cheung et al., 2001), but not all 15 abnormalitiesappeared together in the same patient (Table 1).

The SI of T1DM patients (mean � SD � 7.42 � 1.88;median � 8; mode � 9) differed significantly from theSI of normal control subjects (mean � SD � 0.67 �0.78; median � 0.5; mode � 0; P � 0.0001). A moredetailed analysis of the data revealed that the mostcommon abnormalities found in the T1DM patientswere abnormal vessel diameter, abnormal vessel dis-tribution density (abnormal vessel length per unitarea), abnormal vessel morphometry (beaded vessel),damaged vessel, vessel sludging, vessel tortuosity, ab-normal A:V ratio, box car blood flow phenomenon,and abnormal blood flow velocity (Table 1).

The SI of the patients correlated with their respec-tive HbA1c levels as measured on the day of the study

(mean � SD � 9.18 � 1.57) (Fig. 2). However, it wasalso noted that neither SI nor HbA1c necessarily cor-related with the duration of the disease since diagno-sis of the same patients (Fig. 3).

DISCUSSION

Diabetes mellitus is a vascular disease that affectsover 20 million people in the United States. A search inthe literature reveals an abundance of research on thegenetics, epidemiology, immunology, endocrinology,and physiology of the disease. Despite the fact thatover 80% of diabetic complications arise from cardio-vascular disease and vasculopathy, real-time researchdata on diabetic microangiopathy in vivo are limited.The lack of information is due mainly to the unavail-ability of real-time research tools and methodology tononinvasively quantify microvascular abnormalitiesin human subjects in vivo. In this study, through theuse of CAIM, the in vivo microcirculation of humansubjects could be easily, objectively, noninvasively,and quantitatively studied.

Although the correlation between SI (which reflectsthe severity of microvascular abnormalities) andHbA1c in this 12-patient study was expected, it wassurprising to discover that SI did not necessarily cor-relate with the duration of the disease since diagnosisof the same patients. Three patients who had beendiagnosed with T1DM for only a short duration (2–6years) showed as many microvascular abnormalitiesand as much disease severity as patients who hadbeen diagnosed with T1DM for significantly longerperiods of time (8–14 years). Despite the short dura-tion of the disease since diagnosis, a 12-year-old pa-tient diagnosed with T1DM for only 2 years had a SI of9 and HbA1c of 10.4 while a 16-year-old patient whohad been diagnosed with T1DM for 14 years also hadan identical SI of 9 and a lower HbA1c of 8.7. Theseresults suggest an interesting possibility that micro-vascular abnormalities may have developed in pedi-atric T1DM patients before the onset of clinical detect-able hyperglycemia. However, we understand thatthis suggestion is preliminary in nature as the numberof T1DM patients used in this study was small (n �



12). To clarify this interesting possibility, a follow-upinvestigation involving a much larger patient number(n � 35 for patients with �6 years duration since

diagnosis; n � 35 for patients with �2 years durationsince diagnosis; n � 35 for nondiabetic control sub-jects) has been initiated.

FIG. 1. Microvascular abnormalities in the conjunctival microcirculation in diabetic patients. (A) A frame-captured image of the conjunctivalmicrocirculation in a patient. Note the presence of abnormally large vessels (wide diameter), avascularity, vessel sludging, uneven vesseldistribution (density), and abnormal A:V ratio. (B) A captured image showing abnormal vessel morphometry, vessel sludging, and abnormalvessel distribution in another patient. (C) A captured image of the conjunctival microcirculation showing a damaged vessel (hemorrhaging)and hemosiderin deposits. (D) A typical captured image of the conjunctival microcirculation in a diabetic patient showing the box car bloodflow phenomenon and vessel tortuosity. Note that the box car blood flow phenomenon is the most common microvascular abnormality foundin any diabetic (T1DM and T2DM) patient. Optical magnification, 4.5�.

256 Cheung et al.


This study reveals the presence of microvascularabnormalities in pediatric diabetic patients despitetheir young age and the relatively short durations of

the disease. In addition, this study shows that CAIMrepresents the availability of a useful noninvasiveand quantitative technology for in vivo microvascu-

TABLE 1

Severity Index Computation: Detailed Tabulation of the Presence of Microvascular Abnormalities in the 12 T1DM Patients

Microvascularabnormalities

Patient No

1 2 3 4 5 6 7 8 9 10 11 12

Abnormal vesseldiameter � � � � � � � � � �

Abnormal vesseldistribution � � � � � � � � � � �

Abnormal vesselmorphometry(beaded vessel) � � � � � � �

Thickened vessel wall �Damaged vessel � � � � � � �Distended vessel �Vessel sludging � � � � � � � � � � �Vessel tortuosity � � � � � � �Avascularity (ischemia) ? ? ? ? ? ?Abnormal A:V ratio � � � � � � � � � � �Microaneurysm

(micropool) � �Box car blood flow

phenomenon � � � � � � � � � ? � �Hemosiderin deposit � �Comma sign (blocked

vessel) �Abnormal blood flow

velocity � � � � � �

SEVERITY INDEX 4 9 8 6 7 9 7 8 9 4 9 9

Note. An “�” indicates the confirmed presence of a microvascular abnormality and its presence contributes to the computation of the SI forthe patient. A question mark indicates a questionable or limited presence of an abnormality; this presence does not contribute to thecomputation of the SI.

FIG. 2. A scatter-plot graph showing the relationship between SIand HbA1c in T1DM patients in Table 1.

FIG. 3. A scatter-plot graph showing the relationship between SI andduration of disease since diagnosis in the same T1DM patients in Fig. 2.



lar studies in T1DM as well as other vascular dis-eases.

ACKNOWLEDGMENTS

This study was funded in part by a University of California DavisProfessional Development Award (A.T.W.C.), a gift from the W. G.Gilmore Foundation San Francisco, California (A.T.W.C.), HughEdmondson Research Fellowships in the Department of MedicalPathology, University of California Davis School of Medicine(A.R.P., P.L.D., J.G.), a Howard Hughes Medical Institute S.H.A.R.P.Fellowship (P.L.D.), and a Children’s Health Grant from the LucillePackard Children’s Hospital at Stanford University, Stanford, Cal-ifornia (D.M.W., A.T.W.C.).

REFERENCES

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Cheung, A. T. W., Chen, P. C. Y., and Zweifach, B. W. (1996).Computer-assisted intravital (conjunctival) microscopy. Microcir-culation 3, 82.

Cheung, A. T. W., Chen, P. C. Y., Shannon, C. M., Wiltse, S. L., andWun, T. (1997). Conjunctival microcirculation in sickle cell dis-ease (SCD): A computer-assisted intravital study. Microcirculation4, 164.

Cheung, A. T. W., Perez, R. V., and Chen, P. C. Y. (1999). Improve-ments in diabetic microangiopathy after successful simultaneouspancreas–kidney transplantation (SPK): A computer-assisted in-travital microscopy study on the conjunctival microcirculation.Transplantation 68(7), 927.

Cheung, A. T. W., Ramanujam, S., Greer, D. A., Kumagai, L. F., andAoki, T. T. (2001). Microvascular abnormalities in the bulbarconjunctiva of patient with type 2 diabetes mellitus. Endocr. Pract.7, 358.

Zweifach, B. W. (1995). Microcirculatory homeostasis 1930–1990:Insight into microcirculatory readjustments provided by studieson the peripheral circulatory insufficiency of the shock syndrome.Microcirculation 2, 245.

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microvascular abnormalities in pediatric diabetic patients

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