USE OF OCULAR FLUIDS AND RETINA IN POSTMORTEM ANALYSIS

Rebecca Childers, Laboratory Technician Jennifer McCarthy, Assistant Chemist
Robert Everson, Ph.D. Analytical Chemist,
Laboratory Supervisor Stephen B.Hooser,DVM, Ph.D.,
Veterinary Tozicologist, Head,Toxicology Section

Postmortem analysis for chemical constituents of the blood can be difficult unless the blood is colllected very soon after death and prior to coagulation.  However, ocular fluids (aqueous and vitreous humor) and the retina can be useful for the diagnosis of several pathologic conditions, or exposure to various chemicals, for some time after death has occurred. Vitreous (and in some cases aqueous) humor can be used to estimate the time of death and can be useful as an aid in the diagnosis of renal disease, nitrate poisoning, hypomagnesemicsyndromes, calcium status and salt poisoning (Hanna, et.al., 1990, Lincoln and Lane, 1985, McLaughlin and McLaughlin, 1986, McLaughlin and McLaughlin, 1987).  Retina can be used to diagnose organophosphate poisoning, or to prove livestock exposure to some chemicals such as clenbuterol.

In all species that have been examined (cattle, dogs, swine, and rabbits), urea nitrogen and creatinine concentrations in ocular fluid correlate very closely with serum concentrations for up to 24 hours (8h in rabbits) after death at body temperature (37°C). At room temperature (20 to 24°C) or refrigerated (4°C), they can be stable for longer periods of time. Therefore, postmortem vitreous humor urea nitrogen concentrations can be useful to diagnose antemortem renal disease (Drolet,et.al., 1990, Henke and Demarais, 1992, Lane and Lincoln, 1985, Schoning and Strafuss, 1981).

Increased nitrate concentrations can also be detected in the postmortem vitreous humor for up to 24 hours at room temperature (if the concentrations are very high, they can be diagnostic of nitrate poisoning up to 60h following death.)  This has proven very useful in the diagnosis of cattle found dead from what was later diagnosed to be nitrate poisoning.  The detection of high nitrate concentrations in forage combined with high nitrates in the vitreous humor can lead to a diagnosis of death due to nitrate poisoning(Boermans, 1990).The use of vitreous humor for the diagnosis of hypomagnesemia in cattle has received mixed reviews in various studies that have been performed. In some, vitreous and serum magnesium concentrations have been reported to correspond for 36 to 48h postmortem at 24°C (Lincoln and Lane, 1985). However, a recent study using over 250 cows concluded that serum and vitreous magnesium concentrations did no correlate closely enough to diagnose antemortem changes in serum magnesium (McCoy and Kennedy, 1994).

"Measurement of retina cholinesterase inhibition has been successfully used to diagnose organophosphate exposure up to 24h postmortem...."

Therefore, to diagnose hypomagnesemia in cattle, it would be necessary to combine clinical signs and an appropriate history with vitreous humor magnesium concentrations.

Also, the use of vitreous humor to estimate the antemortem calcium and sodium serum concentrations has had mixed reports. Some studies report that both are stable in the vitreous humor and substantially reflect the serum concentrations, while others dispute this. Therefore, the use of postmortem calcium and sodium concentrations in the vitreous must also be correlated to history and clinical signs.

Vitreous humor has been used to roughly estimate the time of death in several species.  It can be useful up to 48 to 72h postmortem.  This is done by measuring the increase of potassium or phosphorous into the vitreous over time.  Since the amount of release varies with temperature, it is important to report the temperature of the animal during the postmortem time interval (Crowell and Duncan, 1974, Schoning and Strafuss, 1980).

Retina can be useful for postmortem diagnosis of organophosphate poisoning and for evaluation of antemortem exposure to certain illegal drugs such as clenbuterol.  Since it is a neural tissue, retina contains a large amount of cholinesterase which is inhibited by organophosphate insecticides.  This inhibition can be measured in the same manner as it is in brain or blood. Measurement of retina cholinesterase inhibition has been successfully used to diagnose organophosphate exposure up to 24h postmortem (Harlinet.al., 1989, Harlin and Dellinger, 1993).  In addition, the retina can concentrate some drugs such as clenbuterol, and store them for long periods of time. Therefore, retina can be used to evaluate the exposure of livestock to this illegal drug (Biolatti,et.al, 1994).

Sample Collection

Vitreous humor can be collected in large animals with the use of a 16 to 21 ga needle attached to a 10 ml syringe.  In small animals, the smaller gauge needle and a smaller syringe may be used.  The needle is inserted through the sclera 1 cm caudal to the limbus(corneoscleraljunction). The needle is directed ventrad and slightly caudad for about 1 to 2 cm to avoid the lens before aspirating fluid. Usually, several milliliters can be recovered.  Some studies indicate that it is best to centrifuge the vitreous following aspiration, therefore, if possible, centrifuge at approximately 833g for 10 minutes following collection.  The vitreous should then be refrigerated for submission.  If for some reason vitreous humor is not available, then aqueous humor from the anterior chamber can be collected. This is done by inserting a small gauge needle through the cornea into the anterior chamber and aspirating the aqueous into a 3 or 5 ml syringe.

For postmortem analysis of retina, the whole eyeball should be enucleated and submitted frozen.  ADDL personnel will then remove the retina form the interior of the eye.

REFERENCES AVAILABLE UPON REQUEST