Intracranial Transplantation Of Olfactory Ensheathing Cells Prolongs The Survival Of Mutant Of SOD1G93A ALS Rats

ObjectiveTo inject olfactory ensheathing cells into the corona radiate of mutantSOD1G93A ALS rats in order to study the improvement of the onset, diseaseprogress, survival rates and behaviors of the rats and to study the treatmentvalue, its mechanisms and to provide experimental evidences for ALStreatment in clinical practices.Methods1Identification of mutant SOD1G93A transgenic ratsCut a small piece of the tail from the young rats that were born for threeweeks, to abstract the DNA, then use about4μL of the solution to conductPCR. Then the products were subjected to electrophoresis and observed in theimaging system. 2Cultivation of olfactory ensheathing cellsWe use the neonatal“green”rats that were born for7days. Disinfect the wholebody, especially the head, then cut the head through the neck, use anotherknife to take out both ensheathing bulbs, put them into ice-cold DF-12andwash them with normal DF-12solution for three times. Peel the membraneunder anatomy lens. Cut the left parts as small as possible with anophthalmology knife. Move the cutted tissues into15ml centrifuge tube.Digest in trypsin-EDTA. Collect the cells and plate them into poly-L-lysine(PLL)-coated dishes. Cultivate the cells with13%certified fetal bovine serumat37℃for CO2.3Purity identification of olfactory ensheathing cellsWe use two kinds of methods, morphology identification andImmunofluorescence stain identification. Observe the shape under invertedmicroscope. The soma looks like a ball or star with protuberances around.Mark the cell nucleus with Hoechst, meanwhile stain with P75NGFR andS-100. Observe under Laser Scanning Confocal Microscope. Double positivecells comparing with the total cells is the purity of olfactory ensheathing cells.4Transplantation of olfactory ensheathing cellsCell transplantation was performed at90-day rat into both wild-type(WT) rats and transgenic SOD1G93A rats using sterotaxic coordinates. Fix the rats on surgery platform after anesthesia with chloral hydrate anesthesia(4%,12ml/kg). Clear the hair on the head, perform a hole about0.5mmdiameter in the skull at the site about+2mm to the anterior fontanelle,1.5mmon the right side of the midline, then insert glass micropipette into the coronaradiate (about2.75mm depth from the surface).Inject5μl cell suspension(5×105OECs) or DF12medium slowly within10minutes for different groups.All animals were performed benzylpenicillin (10mg/kg/day, subcutaneousinjection) continuing3days after surgery without cyclosporine.5Clinical assessments and Inclined board testsObserve the onset, survival time, weight and do the inclined board testsfrom the90-day after born.6Confocal microscopy and motor neurons countDo histological detection on the fifth week(125day) and the seventhweek(139day) after transplantation respectively on all rats. Anesthetize therats with4%chloral hydrate and perfuse transcardially with normal salinesolution, then change4%paraformaldehyde in0.1M phosphate-bufferedsolution. Isolate the cerebral and lumbar spinal cord (between the end of T13and the beginning of L4, including the lumbar enlargement).7ImmunohistochemistryStained the selected sections with immunostaining for Choline Acetyltransferase (ChAT). Cerebral cortex and spinal cord sections werepermeabilized with0.2%Triton X-100and4%paraformaldehyde, andblocked with1mg/ml bovine serum albumin and0.5%normal goat serum inPBS. The sections were incubated overnight at4with rabbit anti-ChAT(Chemicon, AB5042,1:400). The section was treated with second antibodyconjugated with fluorescent dye: goat anti-rabbit IgG Texas Red, in the darkfor2h at room temperature. Sections were coverslipped with30%glycerinand observed under confocal microscope Digitalized microphotographs ofimmunofluorescent sections were saved. The Texas Red was excited by a DyeLaser at595–605nm and was emitted at620nm. The EGFP was excited byan argon laser at488nm and was emitted at530nm.8Western blotRats were anesthetized and killed with pentobarbital (150mg/kg),perfused with ice-cold PBS from the left ventricle. The cerebral cortex andlumbar spinal cord was removed, frozen and stored at80°C until proteinextraction. Then homogenized in ice-cold lysis buffer (0.32mol/L sucrose,1mmol/L ethylenediaminetetraacetate,5mmol/L Tris-HCl, pH7.4,0.1mmol/Lphenylmethylsulfonyl fluoride,10μmol/L eupepsin,10umol/L pepstatin A,and1mmol/L β-mercaptoethanol). Equal amounts of protein per lane (50μg)were loaded onto an8%polyacrylamide gel and separated by electrophoresis at90V for30min and then120V for1.5-2.5h. Proteins were then transferredto nitrocellulose at200V for2h and the membrane was blocked with5%non-fat dry milk/0.5%Tween-20in Tris-buffered saline for2-2.5h. As aninternal control, the same nitrocellulose was incubated with an antibodyspecifically for β-actin (Santa Cruz,1:1000) after being stripped.Results1The survival and migration of olfactory ensheathing cellsGreen fluorescence olfactory ensheathing cells can be detected threeweeks after transplantation astrocytes in the glial scar. They can survive atleast7weeks without immunosuppressant.2Clinical assessment and behavioral testWe do the Hind limb extension, Rotarod, Gait abnormality and screentests. All the observations were done by a blind observer. The score of16represents a complete loss of motor function. Cell transplantation group had agreat improvement of motor function than the other groups with a significantdifference(P <0.01). This improvement occurs in different behavioral testsaimed at exploring various features of motor activity. Similarly, the weightof the OECs group were heavier than the group of the SOD1G93A and theSOD1G93A＋medium group at120days. There is no significant differencebetween the SOD1G93A and the SOD1G93A＋medium group(P>0.05). 3Motor neuron detectionThe Nissl staining detection of cerebral cortex and lumbar spinal cordwere used to count the number of motor neurons. The transgenic groups had asignificant loss in motor neuron cells (P <0.001). The expression of ChATwith immunehistochemistry staining was the same.4Western blotWestern blot quantification was conducted to evaluate the ChAT proteinlevels in the cerebral cortex and spinal cord between the OEC transplantedand the non-transplanted SOD1G93A rats. The results showed that the proteinlevels of ChAT in both SOD1G93A and Medium SOD1G93A weresignificantly decreased; The ChAT protein was different with this.ConclusionOECs can postpone the onset time of SOD1G93A transgenic rats, prolongthe survival time and improve motor functions. The transplanted OECs cansurvive at least7weeks, and they can protect the motor neural cells fromdying.