title: Blockers of carbonic anhydrase can cause increase of retinal capillary diameter, decrease of extracellular and increase of intracellular pH in rat retinal organ culture.
abstract: BACKGROUND
  At least in normal-pressure glaucoma a vascular genesis with hypoperfusion and regulation impairment is discussed. This may lead to malnutrition of retinal ganglion cells and apoptosis. The retinal microvasculature has a small functional reserve. In addition, the retinal microvessels lack the autonomic nerves that are normally found in other tissues. Thus, no systemic influences reach the retinal capillaries apart from circulating hormones or transmitters. Blockers of carbonic anhydrase (CA) may modulate regional blood flow by mediating changes in extra- and intracellular pH. However, it is still unclear (1) whether blockers of CA really change the pH near the retinal capillaries and (2) how changes in the local pH affect the capillary tone in situ. Therefore, we tested dorzolamide and acetazolamide in our model of the freshly enucleated rat retina.
  
  
  METHODS
  Adult Sprague-Dawley rats (of both sexes, 250-350 g) were killed and retinae were prepared. The retinae were gently separated from the retinal pigmented epithelium and were observed in a chamber for electronic light microscopy or were fixed for immunohistochemistry. Electronic light microscopy of the retinal cells was performed with a Zeiss Axiovert microscope equipped with differential interference contrast (DIC) optics. Changes in capillary diameter were measured using an Openlab acquisition system and analyzed statistically using ANOVA. In addition to light microscopy the intracellular pH was analyzed in the whole mounts by ratio imaging of the pH using the special dye BCECF-AM (2,7 -bis-(2-carboxyethyl)-5-(and -6)-carboxyfluoresceinacetoxymethyl ester) and the extracellular pH using BCECF (2,7 -bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein).
  
  
  RESULTS
  Pericytes of most segments of retinal capillaries are immunoreactive for alpha-smooth muscle actin (SMA). The SMA immunostaining is strong around the nucleus; the endothelial tube is visible by virtue of the slight immunoreactivity of the surrounding pericyte processes. Acetazolamide and dorzolamide showed statistically significant vasoactive effects in retinal capillaries. Vasodilation increased by up to 105% of that in control capillaries after 5, 10 and 15 min. CA inhibitors were found to be able to induce intracellular alkalization in retinal cells. After addition of dorzolamide or acetazolamide the extracellular pH decreased from 7.4 to 7.2 concomitant with diameter changes.
  
  
  CONCLUSIONS
  The tube-like pattern of SMA immunoreactivity demonstrates the presence of contractile elements within the pericyte processes of the rat retina. Thus, pericytes may act as a regulation element within the retinal microcirculation. Our results further suggest that CA inhibitors are able to decrease pH in the extracellular space; however, the pH within the cells increases. The increase in capillary diameter is concomitant with these pH changes. Thus, we may conclude that CA inhibitors can relax pericytes and might improve the retinal blood supply.
authors.affiliation: Institute of Anatomy, Medical Faculty Carl Gustav Carus, TUD, Fetscherstrasse 74, 01307 Dresden, Germany.
authors.forename: Friedemann
authors.initials: F
authors.lastname: Reber
authors.forename: Udo
authors.initials: U
authors.lastname: Gersch
authors.forename: Richard W
authors.initials: RW
authors.lastname: Funk
mesh_terms: D000086:Acetazolamide / Q000494:pharmacology; D000199:Actins / Q000378:metabolism; D000818:Animals; D002196:Capillaries / Q000502:physiology; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology*; D005110:Extracellular Space / Q000502:physiology; D005260:Female; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology; D009924:Organ Culture Techniques; D020286:Pericytes / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000187:drug effects*; D012171:Retinal Vessels / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology; D014664:Vasodilation / Q000502:physiology*
chemical_list: D000199:Actins; D002257:Carbonic Anhydrase Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013449:Sulfonamides; D013876:Thiophenes; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; C062765:dorzolamide; D000086:Acetazolamide