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

In histological studies using retinas, eyes are commonly fixed with aldehyde derivatives administered by immersion or perfusion. However, the histology of rat retinas chemically fixed as a whole eye is typically inferior to the histology of retinas that are immediately fixed after acute dissection from the rest of the eye. Chemical fixation without dissection often results in neuronal swelling resembling excitotoxic damage induced by ischemia because the retina is protected by the sclera and is thus poorly accessible to immersion or perfusion fixation techniques. In order for the acute dissection technique to work properly, it must be completed in a timely manner, which may be difficult under some circumstances. Microwave irradiation is an alternative method for fixing tissues that are inaccessable to chemicals. We examined the effectiveness of microwave irradiation of the whole eye as a substitute for acute retinal dissection. To study the feasibility of microwave 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 ml artificial cerebrospinal fluid (aCSF) and irradiated with a household microwave oven. For morphological comparison, control eyes were immersed in a chemical fixative containing 1% paraformaldehyde and 1.5% glutaraldehyde. All eyes were 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 retinas of chemically fixed whole eyes showed neuronal swelling similar to excitotoxic ischemic damage, suggesting that conventional immersion methods provide poor whole eye fixation. The neuronal degeneration observed with conventional immersion fixation was not found in retinas of whole eyes fixed with 20 sec of microwave irradiation. During microwave irradiation the temperature in the bathing aCSF rose to 55-72 degrees C. In some eyes, overcooking 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 fixation may be preferable for retinal histology if chemical fixation following acute dissection is not feasible.