Abstract:
The External Granular Layer (EGL) of the cerebellar cortex is a region of proliferative cells which migrate to their last position in the internal granular cell layer of the cerebellum. Delay in the transit of cells from the EGL has been associated with deficiency of tryptophan, an amino acid, which is important in cerebellar maturation and in the disappearance of the EGL. The role of tryptophan in the development of the cerebellar cortex has not been fully looked at. In this study, the profile of the EGL in neonatal mice was examined following maternal administration of tryptophan.
Forty pregnant female Swiss mice weighing between 28 and 45g were allotted to four groups (n=10). The day the mice delivered was postnatal day (PND) 0. Control group (Group I) received distilled water, Groups II-IV orally received 100, 200 and 300mg/kg body weight solution of tryptophan, respectively. Dams in each group received a daily dose of tryptophan postpartum for 0,7,14,21 days and the pups (n=5) were sacrificed after the last dose. In the pups, Brain Weight (BW) and Cerebellar Weight (CW) were measured by a weighing scale; Brain Tryptophan (BT) and Serum Tryptophan (ST) levels were measured by spectrophotometry. The cerebella were fixed in 10% formalin, coronal sections taken at the paraflocculus were stained with haematoxylin and eosin for estimation of cerebellar cortical layer width, neuronal cell counts and mitotic index. Other coronal sections were stained with cresyl violet for radially oriented granule cells; modified Golgi stain for dendritic arborization of Bergmann glial cells; ki-67 immunostaining for cell proliferation and Bcl-2 immunostaining for apoptotic cells. Data were analysed using ANOVA at α0.05.
On PND7, BW of GroupII (0.25 ± 0.07g) was significantly greater than Group I(0.20 ± 0.03g), GroupIII (0.20±0.05g) and GroupIV (0.17±0.03g) and CW of GroupII (0.06±0.01g) was greater than GroupI (0.05±0.01), GroupIII (0.05±0.01) and GroupIV (0.03±0.07g). External granular layer width of GroupI (0.11±0.00mm) was less than GroupII (0.13±0.05mm), GroupIII (0.13±0.13mm) and GroupIV (0.13±0.02mm) on PND14. Percentage BT in GroupII (9±2x10-4%) and GroupIII (12±2x10-4%) was significantly higher than GroupI (8±2 x10-4%) and GroupIV (7±2 x10-4%). Serum tryptophan in GroupII (0.020±0.012%) was significantly higher than GroupI (0.005±0.002, GroupIII (0.011±0.005) and GroupIV (0.002±0.001%) on PND21. The EGL density of GroupIV (34.0±5.15mm2) was higher than GroupI (23.6±8.02mm2), GroupII (30.7±2.5mm2) and GroupIII (31.4±2.5mm2). Mitotic index was significantly higher in GroupIV (11.8%) than others (6.6,7.9,6.2%). Number of radially oriented granule cells was significantly higher in GroupII (2.6±0.62; 2.98±1.02), GroupIII (2.9±0.93; 2.3±0.42) and GroupIV (2.7±0.29; 3.1±0.86) than GroupI (1.67±0.48; 1.28±0.43) on PND7 and 14. The EGL persisted in GroupII pups compared to others on PND21 and there were more Bergmann glial cytoplasmic processes in GroupII than in other groups. Percentage positivity of ki-67 cells was significantly increased in GroupII and GroupIII on PND7 and 14. However, the percentage positivity of Bcl-2 was significantly increased in GroupII on PND7.
Low dose tryptophan prevented the early loss of cells in the EGL which can be a potential site for adult neurogenesis and a source of stem cells.
