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NEONATAL PINEALECTOMY IN RAT – OWN EXPERIENCE

To determine the role of the pineal gland and its secretory product melatonin on various aspects of the functioning of the organism the gland can be removed surgically within 18 hours after birth. We performed pinealectomy in deep hypothermia and under the operating microscope. The rats were induced into a state of suspended animation by placing them in the freezing compartment at minus 20 Celsius degrees. The cessation of respiration and heart beat lasted about 15 minutes. At this time the pinealectomy was carried out. In some cases there was a little hemorrhage. We had no mortality after surgery.
Key words: rat, pineal gland, pinealectomy, melatonin.

INTRODUCTION

Since 1933 the pinealectomy in rats has become an excellent model showing the impact of main pineal hormone, melatonin, on physiology and pathology in mammals..

Melatonin ( N-acetyl-5-methoxytryptamine ) is the major neurohormone secreted by the pineal gland ( 1,2 ).

Melatonin has been reported to possess numerous functions, been involved in sleep initiation, vasomotor control, anti-excitatory actions, regulation of mitochondrial functions ( 1, 2 ). Melatonin and its metabolites were found to have also important immunomodulatory and antioxidant properties owning to their direct and indirect antioxidant actions by scavenging free radicals and by upregulating antioxidant pathways ( 1, 3, 4, 5, 6 ). It also plays a role in the regulation and reset of circadian rhythms with involvement in the measurement of day length, an environmental variable used for seasonal timing of reproduction, metabolism and behavior in animal species ( 1,7,8,9 ). Melatonin has capability to regulate leukocyte function and contributes to the control of inflammation in tissues acting as both an activator and inhibitor of the inflammatory and immune responses ( 10, 11, 12, 13) . Melatonin administration increases the proliferative response of rat lymphocytes, increases the number of NK cells, stimulates the release of pro-inflammatory cytokines interleukin 1 ( IL-1 )and tumor necrosis factor ( TNF-alpha ), enhances phagocyres and modulates apoptosis ( 2 ). Melatonin can act on energy metabolism stimulating mitochondrial boogenesis, increasing the efficacy of the electron transport chain mitochondria, thereby limiting electron leakage and free radical generation ( 14, 15 ). Melatonin can also increase mitochondrial glutation levels, leading to protection against free oxygen species ( 16 ).

METHOD

The pinealectomy was carried out on Sprague-Dawley rats within 18 hours after birth. The surgery was performed in deep hypothermia. Each of newborns was placed in open cardboard box and placed in the freeze compartment of a commercial refrigerator at minus 20 Celsius degrees for 8-10 minutes ( 17 ). This resulted in cessation of respiration and heart beat. Animals remain in a state of suspended animation for about 15 minutes.

The operation was performed under an operating microscope at the twenty-fold magnification. The hypothermic animal is placed on a precooled surface . A midline longitudinal incision was done. This cut exposed the occipital and parietal bones of the skull. An U-shaped skull flap was made by incising with a small lancet around the junction of saggital and transverse sinuses. The flap was lifted up and bent posteriorly exposing the pineal gland. Using 0,5 mm injection needle a slit is made into the dura exposing access to the pineal gland which was removed by surgical micro-suction own construction. In some cases there was a little hemorrhage, this was easily controlled by light compression with swab. The bone flap was replaced and the skin sutured using Mersilk 5-0 ( Ethicon ). The wound was sealed with Cutanol spray ( Pharmacia and Upjohn Company ). The animals recovered within 15 minutes. W achieve optimum temperature placing heating lamp ( 75 W ) above the box with animals. Thermometer lying in the box registers 28 Celsius degrees .

For sham operated animals the procedures described is followed, except pineal gland removal.

When the neonates are mobile they are restored to the maternal cage.

Completeness of removal of the pineal gland is verified histologically. No cortical damage or epithalamic damage has been seen as a consequence of this surgery.

DISCUSSION

The pineal gland in rat is a tiny round, rather translucent organ, situated on top of the brain at the confluence of the superior sagittal and transverse sinuses, between cerebral hemispheres an the cerebellum ( 18, 19 ). The pineal gland represents a complex, rather than a single organ in adult rats. One can distinguish pineal tissue in the intercommissural region as a deep pineal, a superficial pineal, which represents the major part of the pineal complex and parenchymal stalk of variable length ( 20 ).

Pinealectomy is known to remove the nocturnal elevation of serum melatonin level and decrease melatonin concentrations. It has been suggested that reduction of serum levels of melatonin after pinealectomy increases oxidative stress in a number of tissues ( 5 ). Melatonin and its lower serum levels after pinealectomy has influence on immunity and aging ( 11, 12, 13 ).

Important anatomical structures as transverse sinus, superior sagittal sinus and the confluence of sinuses should be taken into consideration during pinealectomy.

We had no mortality after surgery. Completeness of pinealectomy should be confirmed by histological examination.

The precision in neonatal pinealectomy is most important, for this reason the operation was performed under an operating microscope.

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