Patricia A. Grimes

Acute reversible cataract induced by xylazine and by ketamine-xylazine anesthesia in rats and mice

Combined administration of ketamine and xylazine is used increasingly for safe, effective anesthesia of small laboratory animals. We found that rats injected systemically with ketamine and xylazine at doses recommended for effective anesthesia developed acute reversible lens opacities. Mice given the same drug doses were similarly affected. Testing of each drug alone demonstrated that xylazine was the causative agent. The appearance of cataract was associated to varying degree with proptosis, suppression of the blink reflex, corneal surface drying, and mydriasis. All of these ocular effects, including cataract also could be induced locally by topical application of xylazine to one eye, with untreated contralateral eyes showing no drug effects. A possible cause of xylazine-induced transient lens opacification is trans-corneal water loss and alteration of aqueous humor composition due to corneal exposure. Additional action on aqueous humor formation and the lens itself may be due to the alpha-2-adrenoceptor nature of xylazine. Whatever the cause of cataract induction, the occurrence of this phenomenon during ketamine-xylazine anesthesia appears to be associated with marked changes in the physiological state of the eye. Therefore, the side-effects of anesthetic drug combination should be considered prior to its use on animals for studies of ocular physiology.

Early morphological alteration of the pigment epithelium in streptozotocin-induced diabetes: Increased surface area of the basal cell membrane

We have observed a marked increase of membrane infolding at the basal surface of pigment epithelial cells in streptozotocin-diabetic rats after three to five weeks of hyperglycemia. The extent of basal infolding was determined by stereological analysis of electron micrographs. These measurements demonstrated that in 12 diabetic eyes, the average length of basal cell membrane per unit length of Bruchs membrane was 33% greater than in an equal number of control eyes (P < 0·001). The linear increase represents an approximate doubling of the area of the basal cell surface. This structural modification may be associated with the altered permeability of the cell layer indicated by our previous finding of abnormal penetration of fluorescein into the pigment epithelium in diabetic rats.

Autonomic neurons supplying the rat eye and the intraorbital distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactivity

We traced the origin and path of autonomic nerves to the rat eye using, as an aid to dissection, a modified thiocholine method for the histochemical demonstration of cholinesterase. When applied to whole nerves and ganglia supplying the rat eye, this procedure is not specific for cholinergic neurons; instead it stains both sympathetic and parasympathetic nerves, many of which are otherwise too fine to identify in dissection. We found that nerves from the superior cervical and pterygopalatine ganglia form a plexus at the orbital apex corresponding to the retro-orbital plexus found in rabbit, monkey and man. In the rat, nerves from the retro-orbital plexus travel peripherally to the superior surface of the optic-nerve sheath. Here, they fuse with long ciliary nerves and the post-ganglionic nerves from the ciliary ganglion to form another dense nerve-fiber plexus that ultimately supplies the eye. Importantly, the plexus on the optic nerve contains many isolated or aggregated ganglion cells. These are comparable in number to those in the ciliary ganglion itself and are assumed to be accessory ciliary neurons. Using immunohistochemistry, we also sought evidence for vasoactive intestinal polypeptide in these ganglia and nerves. As previously known, many pterygopalatine ganglion cells are immunoreactive for this peptide. Vasoactive intestinal polypeptide (VIP)-like immunoreactive nerve fibers are present in nerves from the retro-orbital plexus to the optic-nerve sheath plexus, in most nerves of the latter plexus, and in most nerves entering the eye. Furthermore, a small proportion of nerve cells in the main and accessory ciliary ganglia also are immunoreactive for VIP. We conclude that in addition to the pterygopalatine ganglion, the ciliary ganglion and its accessory ganglia are sources of VIP-like immunoreactive nerves in the rat eye.

Nitric oxide synthase in autonomic innervation of the cat carotid body

In the cat carotid body, nitric oxide synthase (NOS) immunoreactivity and NADPH diaphorase activity localize in nerve fibers mainly associated with blood vessels and occasionally lying close to glomus cells. The NOS-positive innervation originates in part from multipolar ganglion cells scattered in and around the carotid body and in the glossopharyngeal nerve. In the superior cervical ganglion, NOS and diaphorase staining localizes to many preganglionic axons and also to a small population of vasoactive intestinal peptide-positive, presumably cholinergic, ganglion cells. Positively stained ganglion cells are absent in the petrosal ganglion and very rare in the nodose ganglion, although both sensory ganglia display characteristic distributions of cells immunoreactive for calcitonin gene-related peptide, substance P and tyrosine hydroxylase. The NOS-positive innervation of the carotid body thus appears to be autonomic, originating mainly from a population of dispersed ganglion cells, and probably parasympathetic in nature. The superior cervical ganglion also may supply some pre- or postganglionic NOS-positive axons. Nitric oxide released from these nerves could affect glomus cell activity directly or indirectly by vasoregulation.

Nitric Oxide Synthase Occurs in Neurons and Nerve Fibers of the Carotid Body

Nitric oxide (NO), a free radical, can be produced by vascular endothelial cells and certain neurons of the central and peripheral nervous systems upon activation of its biosynthetic enzyme, nitric oxide synthase (NOS). NO may function as a non-conventional neurotransmitter in the nervous system, and either NO or an NO-producing compound is believed to be the endothelium-derived relaxing factor of the vascular system. An antiserum recognizing the molecular form of NOS present in brain has been used to localize the enzyme in brain and peripheral tissues (Bredt et al., 1990). NOS containing neurons also display intense NADPH-diaphorase activity (Dawson et al., 1991; Hope et al., 1991), and this histochemical reaction, though less specific than immunohistochemistry, provides a label for NOS activity in both central and peripheral neurons.

The mouse Cat4 locus maps to Chromosome 8 and mutants express lens-corneal adhesion

Cat4 is the second largest allelism group in the collection of mouse dominant eye mutations recovered in Neuherberg and carriers express anterior polar cataract, central corneal opacity, and lens-corneal adhesions. We have mapped the Cat4 locus of the mouse to central Chromosome (Chr) 8 at position cM 31. Histological characterization of Cat4(a) heterozygotes and homozygotes indicates failure of separation of the lens vesicle from the surface ectoderm. Human anterior segment ocular dysgenesis (ASOD) is autosomal dominant, carriers express an eye phenotype similar to that of Cat4(a) carriers, and it has been mapped to a region of 4q homologous to mouse central Chr 8. Thus, on the basis of phenotype and map position, Cat4 may be a mouse model of human ASOD. The genes Junb, Jund1, Mel, and Zfp42 are discussed as possible candidates for Cat4.

Peptide immunoreactivity of the ciliary ganglion and its accessory cells in the rat

By means of immunohistochemistry, calcitonin gene-related peptide, Leu-enkephalin and neuropeptide Y localize to rat ciliary and accessory ganglion cells. The proportion of ciliary and accessory neurons immunoreactive to each peptide is provided and compared to previous data for vasoactive intestinal peptide. These findings indicate considerable neurochemical complexity for a parasympathetic ganglion with a small cell population.

Neoplastic transformation in vitro of hamster lens epithelium by simian virus 40.

Hamster lens epithelium infected with simian virus 40 underwent transformation in vitro and produced tumors when injected into homologous hosts. Undisturbed lens epithelium in man and experimental animals has not been observed to undergo neoplastic change. The virus-induced tumors contained undifferentiated cells that were either polygonal or spindle-shaped. Their origin from lens epithelium seems certain since it is possible to isolate this unique structure free of connective tissue and blood vessels.

Genetic mapping of a mouse ocular malformation locus, tcm, to chromosome 4

The Tcm mutation in the mouse is an autosomal dominant ocular malformation manifesting as microphthalmia, iris dysplasia, cataract, and coloboma. As a first step to cloning the Tcm gene, we report the localization of the Tcm mutation with respect to known microsatellite markers. Backcross progeny carrying the Tcm mutation were produced by mating Tcm/+ heterozygous mice to normal C57BL/6 partners. Genomic DNA from each mouse was subjected to PCR analysis to identify simple sequence length polymorphisms. Our results locate Tcm to Chr 4 and suggest candidate genes responsible for the Tcm phenotype. Finally, ocular histopathology was done in 3-week-old animals to define the extent of the malformation.

Increase of basal cell membrane area of the retinal pigment epithelium in experimental diabetes.

Stereological analysis of electron micrographs of the pigment epithelium of rats with drug-induced diabetes demonstrated an increase of plasma membrane surface area at the basal aspect of the cells. In none of the diabetic animals examined was there any evidence of breakdown of the blood-retinal barrier to the protein tracer, horseradish peroxidase. Statistically significant increases in basal plasma membrane length and surface density (surface area per unit cell volume) were measured in both streptozotocin and alloxan-injected rats after four weeks of diabetes. When hyperglycemia in streptozotocin-injected rats was promptly reversed by transplantation of normal pancreatic islets, the increase of membrane surface area did not occur. We conclude, therefore, that increased basal surface area of pigment epithelial cells is related to the diabetic condition rather than to a toxic action of the diabetogenic agents. Furthermore, increased membrane surface area was present in streptozotocin-diabetic rats killed after six months of diabetes indicating that the structural change is relatively stable. Relation of basal membrane alteration in the pigment epithelium to any functional disturbance of the barrier cell layer or of the retina in diabetes remains to be established.