Acetylcholinesterase activity in the brain of alloxan diabetic albino rats: Presence of an inhibitor of this enzyme activity in the cerebral extract

Background and Aim: Ischemic manifestations and cerebral dysfunction have been demonstrated in diabetes. However, the pathogenesis of diabetesinduced cerebral dysfunction still remains to be elucidated. Hence, the present study was initiated. Materials and Methods: Type-2 diabetes was induced in albino rats (280–300g) with alloxan monohydrate (40 mg/Kg i.v.,) and the cerebrum, cerebellum and medulla oblongata of the brain were used 48 h after alloxan injection for modulations in acetylcholinesterase (AChE, EC 3.1.1.7) activity. Results: AChE activity in the discrete regions of the brain of rats decreased signifi cantly (P<0.01, 0.05 and 0.05 respectively) in diabetes. In vitro studies using cerebral extract from alloxan diabetic rats demonstrated signifi cant (P<0.05) inhibition of AChE activity in the brain of normal animals. Feeding with Cichorium intybus (chicory) leaf extract (500 mg/Kg) for 10 days resulted in an increase in AChE activity. Conclusion: The impairment in the glycemic control is the basic mechanism causing inhibition of neuronal activity. Cerebral extract from alloxan diabetic rats signifi cantly inhibited the brain AChE activity of normal animals, indicating the presence of an inhibiting factor in the cerebrum of diabetic rats. Cichorium intybus when fed for 10 days offered neuroprotection by stimulating AChE activity.


Introduction
Diabetes mellitus is a metabolic disorder with a potential to cause cerebral injury. [1,2] In diabetic neuropathy, it is known that cerebral uptake of glucose is signiÞ cantly aff ected [3][4][5] and the brain suff ers from hypoglycemic episodes. [6] Diabetes-induced hyperglycemia is known to increase the extent of neurological disorders due to inhibition of enzymatic activities connected to neurotransmission [7,8] in the CNS of mammals and the changes observed have been att ributed to alterations occurring in the levels of RNA and proteins. [9] Although the ischemic manifestations and cerebral disorders in diabetes have been documented, the pathogenesis of diabetes-induced cerebral dysfunction still remains to be elucidated. In view of this, the present study was initiated to demonstrate cerebral manifestations of diabetes as in diabetes brain suff ers from recurrent episodes of hypoglycemia and impaired glucose oxidation leading to impaired neuronal activity. Eff orts have also been made to investigate the neuroprotection off ered by Cichorium intybus (Chicory) in view of its cardioprotective [10] eff ects reported earlier.

Materials and Methods
Male albino rats of Wistar strain (280-300 g) were maintained in polypropylene cages (3 in each cage at 26°C; 12 h light:dark cycle). The animals were fed standard pellet diet and water ad libitum. The rats were rendered diabetic with alloxan monohydrate (40 mg/kg; i.v.) and used 48 h aft er alloxanization. Animals with 220 mg/100 ml blood sugar level were considered diabetic and utilized for experimentation.
The study protocol was approved by the Institutional Animal Ethics committ ee.
The rats were divided into the following groups: Gr I: Controls Gr II: Diabetic Gr III: Diabetic rats fed on dried leaf powder of Cichorium intybus (500 mg/kg) for 10 days.
The animals were killed by decapitation and the brains were quickly removed and washed in ice-cold saline. Different regions of the brain were separated with sterilized Þ ne bent forceps and scalpel, weighed in an electric balance in mammalian Ringer and immediately used for the determination of acetylcholinesterase (AChE) activity.
Chemicals and reagents: Alloxan monohydrate was obtained from Sigma Chemicals (St. Louis, MO, USA). All other chemicals used were of analytical grade. Double distilled water was used for biochemical assays.
Homogenate (10% w/v) of diff erent regions of the brain was prepared in ice-cold 0.02 M phosphate buff er (pH 7). The homogenate was centrifuged for 20 min at 6000 rpm and the supernatant was used for the assay. Activity levels of AChE were determined spectrophotometrically by the method of Hestrin. [11] Eff ects of in vitro administration of the aqueous cerebral extract (0.5 ml) from alloxan diabetic rats on the activity level of brain AChE of normal animal were studied. It was noted from preliminary experiments that only the cerebral extract from the diabetic animal was capable of causing signiÞ cant decrease in enzyme activity of brain in normal animals and therefore the eff ects of in vitro administration of the extracts from cerebellum, medulla and whole brain of diabetic animals were not studied.
The assay systems, on the addition of aqueous extract of the brain from normal animals constituted controls. Preliminary experiments indicated negligible diff erence between the activity levels of controls prepared by the addition of cerebral extract from diabetic animals to the assay system aft er terminating the enzyme reaction and those receiving the extract of the brain from normal animals. Hence, the latt er was used as the experimental controls. The assay systems with the aqueous cerebral extract from the diabetic rat were the experimental samples.

Studies on neuroprotection by intervention with Cichorium intybus
Cichorium intybus: (Kasni-Hindi; Hindba Arabic and Chicory-English. The plant has utility in Indian (Unani) system of medicine for the maintenance of blood glucose level in diabetes and treatment of liver and kidney ailments.

Plant Material:
Fresh Cichorium intybus Leaves were collected from chicory farm in Vaniyambadi, Tamilnadu, India and shade dried for 21 days. The dried leaves were Þ nely powdered and kept in air-tight glass containers before use. The plant was authenticated by a botanist, Bangalore University, Bangalore.
Cichorium intybus: (Dried leaf powder 500 mg/kg body weight-fed rats (for 10 days) were rendered diabetic and used 48 h aft er alloxanizatioun to demonstrate the neuroprotection by investigating AChE activity in the brain. For this, AChE isozymic spectrum was resolved by polyacrylamide gel electrophoresis and compared with that of control and diabetic patt ern in the cerebral cortex. 50% (w/v) homogenates of the cerebral cortex were prepared in deionized distilled water and centrifuged at 7000 rpm for 1 h at 4° C. Ten µl of the supernatant was used for the electrophoretic run. The gels were run at 6 m Amps/tube (D.C. power supply) for 2 h using Tris-Boric acid-EDTA buff er at pH 8.5.
Aft er the run, the gels were cut into 2 mm bits and each bit was homogenized in 1 ml of phosphate buff er (pH 7.0) centrifuged and used immediately for AChE determination as described above [11] and the patt ern presented graphically.

Results
The weight of the animal and its brain exhibited considerable decrease as a function of the disease [ Table  1]. The blood sugar level demonstrated 120.5% elevation as a function of the disease [ Table 1].
From the values given in Table 2, it is clear that the activity level of AChE was decreased in the cerebrum, cerebellum and medulla oblongata from diabetic rats. As the animals were experimented 48 h aft er alloxan administration, the observed decline in enzyme activity is related to early stage of alloxan diabetes. It is also seen [ Table 2] that the decrease in the level of AChE activity showed variation in relation to the region of the brain. The activity level of AChE was higher in the cerebrum of both normal and alloxan diabetic rats compared to the other regions of the brain. Further, contrary to expectation, the cerebrum showed marked response for the change in the activity level of AChE and not the brain stem [ Table 2].

Discussion
The decrease in the activity level of AChE in diabetes observed may be a reß ection of the type of electrical change in the nervous tissue on inducing alloxan diabetes. Such a decrease in the activity levels of AChE in diff erent regions of the brain of alloxan diabetic rats may be either due to the inhibition of the enzyme synthesis by the altered cellular environment prevailing in the brain, or due to a decrease in the rate of enzyme synthesis. Earlier studies also indicated the inhibition of AChE synthesis in the CNS of rats with alloxan diabetes. It is suggested that the cause of inhibition of AChE synthesis is inadequate permeation of glucose across the surface membrane during diabetes.
Activity levels of AChE in the cerebrum, cerebellum and medulla oblongata of normal animals determined in the presence of the cerebral extract from diabetic animals were signiÞ cantly low [ Table 3, Figure 1]. The    changes produced by the nervous extract of diabetic rats indicated the presence of an inhibiting substance/s in the cerebral extract of alloxan diabetic rats capable of depressing the AChE activity of normal animal on in vitro administration.
From the isozymic specrum presented in Fig 2, it is clear that the decline found in the activity of AChE is due to abolition of an isozymal form during diabetic state. Characteristically, in the diabetic rats (fed with dried leaf powder of Cichorium intybus), the isozymic patt ern returned to the control (Gr 1) patt ern with 2 isozymes. This restored neuronal effi ciency and off ered neuroprotection.