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Effect of Cistanche Desertica Polysaccharides on Learning and Memory Functions and Ultrastructure of Cerebral Neurons in Experimental Aging Mice

SUN Yun et al

ABSTRACT Objective To observe the effects of Cistanche desertica polysaccharides (CDP) on the learning and memory functions and cerebral ultrastructure in experimental aging mice. Methods: CDP (Cistanche desertica polysaccharides) was administrated intragastrically 50 or 100 mg/kg per day for 64 successive days to experimental aging model mice induced by D-galactose, then the learning and memory functions of mice were estimated by step-down test and Y-maze test； organelles of brain tissue and cerebral ultrastructure were observed by transmission electron microscope and physical strength was determined by swimming test. Results: CDP could obviously enhance the learning and memory functions (FV0. 01) and prolong the swimming time (P<0. 05), de­crease the number of lipofuscins and slow down the degeneration of mitochondria in neurons(P<0.05), and improve the degeneration of cerebral ultra-structure in aging mice. Qmdusion: CDP (Cistanche desertica polysaccharides) could improve the im­paired physiological function and alleviate cerebral morphological change in experimental aging mice.

KEYWORDS aging, learning and memory functions, Cistanche desertica polysaccharides, ultra-struc­ture, D-galactose

Cistanche desertica, a herbal medicine applauded as 'ginseng from the desert'， has the special effect in reinforcing bone and Marrow and prolonging life span, and its anti-aging effect has been confirmed by mod­ern pharmacological studies"). The natural aging process begins with learning and memory degradation, .followed by the de­generative changes in biological and morpho­logical structures. In this article, D-galac­tose induced aging mouse model was used to observe the effect of Cistanche desertica pol­ysaccharides (CDP) on learning and memory functions and physical strength as well as the ultra-structure of brain tissues. The re­sults were reported as follows.

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METHODS

Animal

ICR mice, 2 months old, of the clear glass, half male and half female, weighing 20 — 22g, were purchased from Experimen­tal Animal Center of Yangzhou University (Certification No. 98002). Ten mice were kept in every chamber, and all the animals were free to access food and water.

Reagents

D-galactose, purchased from Shanghai No. 2 Pharmaceutical Factory, was com­pounded with normal saline to 5 g/L and re­served in the refrigerator at 4°C. CDP (Cistanche desertica polysaccharides) was pro­vided by Nanjing Institute of Wild Plant, u­sing distilled water to compound it to 2.5 g/ L and 5. 0 g/L. Step-down kit and Y-maze apparatus were the products of Shanghai Haodun Medical Apparatus Company.

Aging Model Duplication and Experimental Design1,3,

ICR mice were randomly divided into 4 groups: the control group ( the normal mice), the aging model group; the high dose CDP (Cistanche desertica polysaccharides) group, and the low dose CDP (Cistanche desertica polysaccharides)group, 55 mice in each group. For the con­trol group, 10 ml - kg-1. d_1 normal saline was injected subcutaneously to each mouse. As for the other groups, 50 mg. kg-1 - d_1 D-galactose was hypodermically injected into each animal for 42 days to duplicate the ag­ing model. From the beginning of duplica­tion, 20 ml - kg-1 - d-1 normal saline was ad­ministrated intragastrically to each animal of the aging model group, and 50 mg • kg-1 • d_1and 100 mg - kg-1 - d-1 CDP (Cistanche desertica polysaccharides) were admin­istrated intragastrically to each mouse in low dose CDP (Cistanche desertica polysaccharides) group (Treatment group I ) and high dose CDP (Cistanche desertica polysaccharides) group (Treatment group U ) respectively for 64 days.

Six hrs after the last medication, 30 mice were taken from each group respective­ly to test the learning and memory functions by step-down test and Y-maze test, 15 for each test. The swimming test for evaluating the physical tolerance of mice was conducted 2 hrs after the last medication with 15 mice from each group. Besides, 10 mice from the control group, the model group, and the treatment group H respectively were killed at the end of the experiment for histo-morphological examination.

Step-down Test ⑷

Mice were put in the response chamber to adapt to the environment for 3 min, then the training was started by connecting the e­lectricity to the chamber. The mouse was safe when it was on the platform, but when it jumped to the bottom of the chamber (a mistake), the 36 V electric stimulation would force the mouse to jump back to the platform. The training underwent for 5 min, the accumulative number of mistake­making times within 5 min was recorded. The formal test was carried out 24 hrs later to test the capacity of the mouse on seeking a safe area to escape from electric stimulation. The mouse was put on the platform, and the la­tent time before the first mistake (time of stay at the platform) and the accumulative number of mistake-making times within 5 min was recorded.

Y-maze Test

The Y-maze is a maze box with three chambers arranged radially with the bottom made of copper bars and opening into each other. A lamp is set on the top and an elec­tric source is linked to the bottom of each chamber. The lamp will be lit when the electric circuit is broken, and then the chamber is a safe area to the animal. Con- trarily, when the electric circuit is ener­gized, the darkened chamber becomes an e­lectric stimulation area, which will cause e­lectric damage to the animal. The mouse was first placed into the Y-maze for 2 min for adap­ting itself to the environment, then the circuit (36 —40 V) at the chamber in which the mouse stayed was connected to force the mouse to escape to the safe area. The training was re­peated 15 times to consolidate the light-de­pending safe area memory in the mouse. Then the number of correct and incorrect times of 10 tests were recorded to estimate the correct rate.

Swimming Test(1)

The mouse was placed into a container, which was 28 cm high, and filled with wa­ter, at 15°C in temperature, and it would swim in water till it got exhausted and drowned. The time from putting it into wa­ter to its submerging was counted as swim­ming time, which was used to evaluate the physical tolerance of the mouse.

Sample Preparation of Histo-morphological E­valuation ⑶

Ten mice taken from the control group, the aging model group and Treatment group II respectively were decapitated and the cor­tex of the left cerebrum of each mouse was taken out, doubly fixed by 4% glutaral and 1% osmic acid, dehydrated gradiently by ac­etone, embedded by Epon 812, stained by Pb-U staining. The major organelles were observed under transmission electron micro­scope (TEM, Hitachi H-300, Japan).

Statistical Analysis

The data were expressed as x +s. Sta­tistical comparisons of the results were per­formed with paired t-test.

RESULTS

Effects of CDP (Cistanche desertica polysaccharides) on Learning and Memory Func­tions

The step-down test showed learning and memory functions of aging mice decreased more significantly as compared with those of the control group, manifesting as shortened latency time, and increased accumulative mistake-making times. The latency time pro­longed, and accumulative mistake-making times decreased in treatment group I and U; a significant difference was found when com­pared with the aging model group. The re­sults showed that CDP (Cistanche desertica polysaccharides) could obviously im­prove the learning and memory functions. See Table 1.

Results of the Y-maze test were similar to that of the step-down test, which showed that the rate of correct safety memory was reduced in aging mice. A significant difference existed when treatment groups I and H were com­pared with the model group, suggesting the rate of correct safety memory raised after CDP (Cistanche desertica polysaccharides) treatment.

Effect of CDP (Cistanche desertica polysaccharides) on Physical Tolerance

The swimming time in aging mice was shortened significantly as compared with that in the control group. It was shown that only a high dose of CDP (Cistanche desertica polysaccharides) could prolong significantly the swimming time, thus, have the anti-fa­tigue function. See Table 2.

Effect of CDP (Cistanche desertica polysaccharides) on Organelles in Cerebral Neu­rons

Under an electron microscope, 10 neurons of each sample were observed, and the num­ber of organelle was measured⑷. Results showed that levels of lipofuscin and lysosome increased in neurons, the number of mito­chondria and Golgi complex decreased in the ag­ing model group. The two criteria were re­stored to a certain degree after large dose CDP (Cistanche desertica polysaccharides) treatment. See Table 3.

Effect of CDP (Cistanche desertica polysaccharides) on Brain Tissue Ultra-structure

It was shown that mitochondrial crista broken down and sparsely distributed with vacuolar degeneration. The number of NissP s body was reduced, and degranula­tion appeared in a few of rough surface en­doplasmic reticulum, glycogen granule got reduced, flattened saccule of Golgi complex slightly dilated, with synapse number re­duced, and the number of synaptic vesicle in presynaptic component reduced. See Fig 1.

Fig 1 The mitochondria swelled and its synapse disappeared in neurons. Aging model groups 10,000

The degenerative changes got slightly alleviated after CDP (Cistanche desertica polysaccharides) treatment with obvious­ly increased nucleopores, NissPs bodies and processes, and reduced lipofuscin. See Fig 2 and 3.

Fig 2 Nissl~s bodies of neurons reappeared and some lipofuscin can still be seen. Treatment group II, X 10,000

Fig 3 The nuclear pore increased and nucleolus can be seen in neurons, the cytoplasm is rich. Treatment group K, X 10,000

DISCUSSION

The mechanism of E>-galactose in indu­cing aging is based on the free radical theory of aging. As a result, D-galactose could in­crease free radical content in organisms and impair organelle functions through oxidation damage. Mitochondria as the “energy facto­ry" of cells, can produce lots of free radicals in the respiratory process, and it could be easily attacked by these free radicals. Mito­chondria of an aged organism is easily damaged by free radicals, the damage could not be re­paired due to lack of repair capacity。), and would be aggravated increasingly until ulti­mate death. Lipofuscin is an important mark of aging, its production is closely related to free radical damage, for this reason, it is often used as a criterion in evaluating the level of aging.

Results of this study showed that CDP (Cistanche desertica polysaccharides) in high doses could enhance physical toler­ance in aging mice and improve their learn­ing and memory functions, and alleviate the damage level of cerebral ultra-structure. All these results are in coincidence with our pre­vious research (1,2) and indicated that CDP (Cistanche desertica polysaccharides) can prolong life span, its anti-aging effect may be related to its anti-oxidant capabili­ty and free radicals scavenging effects⑵ to stabilize the membrane system and keep a stable intracellular environment.

From: 'Effect of Cistanche Desertica Polysaccharides on Learning and Memory Functions and Ultrastructure of Cerebral Neurons in Experimental Aging Mice' by SUN Yun et al

---CJIM2001;7(4) =288--292

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