TRAUMATIC BRAIN INJURY Traumatic Brain Injury (TBI) is an injury where a person hits their head on object,resulting in serious brain damage. TBI is often referred to as a silent Epidemic or the unseen injury Because victims generally look fine

incidence TBI annually affects about 1.5 million people in the USA.5.3 million people are living today with disability related to TBI.(2005) In Europe the annual incidence of fetality or admission to hospital owing to TBI is estimated to be 235 per population.(2010) In Iran 993 cases of head injury during the two years has occurred(73% male and 27% women).2000

mesenchymal bone marrow

APPEALING CHARACTERSITICS  Multiipotent - self renewal and wide differentiation into multiple lineages  Can be incorporated direct into other tissues  Low immunogenicity and suppress alloreactive T cell response (transplanted allogenic MSC are not rejected)  Easy to obtain Regenerative medicine and Therapeutic issues

We reported that MSCs administered intravenously after TBI enter rat brain and express the neuronal cell phenotypes bromodeoxyuridine(BrdU) was employed as the marker of donor Cells. BrdU incorporated into the DNA of MSCs can be diluted when the cells proliferate and the signals are lost. A complementary deoxyribonucleic acid (cDNA) probe that exhibits specificity for the rat Y chromosome was generated by using a sequence specific to the murine Sry gene, the sex-determining region of the Y chromosome.

MATERIALS AND METHODS Preparation of male rat MSCs: MSCs were harvested from 24 male Wistar rats. the cells were incubated for 3 days. Culture medium is Iscove’s modified Dulbecco’s medium. Approximately 2×10 ⁶ cells in 300 µl of saline were injected into the tail vein of each female rat.

Animal models controlled cortical impact device was used to induce the injury. Injury was induced by impacting the left Cortex (ipsilateral cortex) with a pneumatic piston containing a 6-mm diameter tip at a rate of 4 m/s

Animal models Group 1 rats (n=4), MSCs were injected into the tail vein 24 hours after TBI and the rats were killed 8 days after brain injury. Group 2 rats (n=4), received the same conditions as Group 1, but were killed 15 days after TBI Group 3 rats (n=4), received saline via the tail vein 24 hours after TBI and were killed 15 days after TBI Group 4 rats (n=4), received TBI and were killed 15 days after TBI

Generation of the cDNA probe specific to rat Y chromosome by polymerase chain reaction The 459-base pair product was obtained using a primer set with the following sequence: 5' primer, 5'-AGATCTTGATTTTTAGTGTTC-3' and 3'primer, 5'-TGCAGCTCTACTCCAGTCTTG-3' to regions of the Sry gene of the murine “sex- determining region” carried on the Y chromosome.

analysis under light and fluorescent microscops A section of each block of the brain and other organs was stained with hematoxylin and eosin for morphological analysis under light microscop. Brain sections were initially immunostained with diaminobenzidine (DAB) for detection of a neuronal marker, NeuN, and an astrocytic marker, glial fibrillary acidic protein (GFAP). Subsequently, in situ hybridization was performed on the same section with fluorescence labeling.

Neurological function evaluation  NSS: is a composite of the motor (muscle status, abnormal movement),sensory (visual, tactile and proprioceptive) and reflex tests  rotarod motor test: The tests were measured on all rats preinjury and on day 1, 4, 7 and 14 after MSC administration

RESULTS Distribution and identification of Y chromosome positive cells: Numerous Y chromosome-positive cells were found in the vessels of the brain, heart, lung, liver, kidney, muscle, spleen,and bone marrow of the female rats receiving the intravenous infusion of MSCs obtained from male rats No Y chromosome-positive cells were observed in the slides from the group with TBI or the group with TBI+saline

In brain, Y chromosome-positive cells were detected in the boundary zone of the injured area, the striatum, the cortex, and the corpus callosum of the ipsilateral hemisphere.

The numbers of Y choromosome-positive cell in the ipsilateral hemisphere were greater than those in the contralateral hemisphere.

These data indicate that the intravenous administration of MSCs 24 hours after TBI reduces the motor and neurological deficits after TBI.

DISCUSSION  male rat MSCs injected intravenously enter the brain and reduce motor and neurological deficits by Day 14.  some of the cells migrating into the parenchyma of the brain express the neuronal marker and the astrocytic marker.  At Day 14 after MSC treatment, Y chromosome- positive cells were significantly greater than on Day 7  Many more Y chromosome-positive cells found in the female rat in this study than Brdu-positive cells in our previous study.  these cells are also present in other organs and did not cause any obvious adverse effects.

The mechanisms responsible for MSC engraftment into brain MSCs may enter brain from blood brain barrier disruption or in response to signals from cytokines and cell surface receptors. MSCs produce an array of trophic factors cytokines and other neural restorative factors. The cytokines produced by MSCs may directly promote tissue plasticity or stimulate glial cells to produce neurotrophic growth factors such as brain- derived neurotrophic factor and nerve growth factor.