Comparison of rat retinal fixation techniques: Chemical fixation and microwave irradiation

In histological studies using retinas, eyes are commonly fixed with aldehyd e derivatives administered by immersion or perfusion. However, the histolog y of rat retinas chemically fixed as a whole eye is typically inferior to t he histology of retinas that are immediately fixed after acute dissection f rom the rest of the eye. Chemical fixation without dissection often results in neuronal swelling resembling excitotoxic damage induced by ischemia bec ause the retina is protected by the sclera and is thus poorly accessible to immersion or perfusion fixation techniques. In order for the acute dissect ion technique to work properly, it must be completed in a timely manner, wh ich may be difficult under some circumstances. Microwave irradiation is an alternative method for fixing tissues that are inaccessable to chemicals. W e examined the effectiveness of microwave irradiation of the whole eye as a substitute for acute retinal dissection. To study the feasibility of micro wave methods, we compared retinal morphology using microwave irradiation to morphology using conventional immersion fixation methods. Eyes were removed from rats, placed in a container with 2 or 20 mi artifici al cerebrospinal fluid (aCSF) and irradiated with a household microwave ove n. For morphological comparison, control eyes were immersed in a chemical f ixative containing 1% paraformaldehyde and 1.5% glutaraldehyde. All eyes we re embedded in araldite for evaluation by light microscopy. Retinal segments acutely isolated before immersion fixation revealed intact histology whereas retinal segments exposed to 60 min of simulated ischemia showed severe neuronal degeneration. Using an immersion technique, the ret inas of chemically fixed whole eyes showed neuronal swelling similar to exc itotoxic ischemic damage, suggesting that conventional immersion methods pr ovide poor whole eye fiuation. The neuronal degeneration observed with conv entional immersion fixation was not found in retinas of whole eyes fixed wi th 20 sec of microwave irradiation. During microwave irradiation the temper ature in the bathing aCSF rose to 55-72 degrees C. In some eyes, overcookin g produced chromatin clumping and a small loss of contrast in staining. Although nuclear clumping and diminished staining occasionally result from overcooking, ischemic damage is well controlled with microwave fixation of enucleated eyes. When the optimal conditions are defined, microwave fixatio n may be preferable for retinal histology if chemical fixation following ac ute dissection is not feasible. (C) 2000 Academic Press.