| Amyotrophic lateral sclerosis(ALS)is a midlife-onset neurodegenerative disorder that leads to death within 3-5 years after diagnosis.The selective motoneuron degeneration in the motor cortex,brainstem and spinal cord results in muscular weakness and paralysis.Although more than 90% of cases are sporadic,Cu2+/Zn2+ superoxide dismutase(SOD1)gene mutations are the most well-known form in familial ALS.And the transgenic mice over-expressing the mutant human SOD1-G93 A gene have been widely used as a tool in seeking effective treatment method and revealing the pathogenesis of ALS.Vascular endothelial growth factor(VEGF)is regarded as an important factor in angiogenesis under physiological and pathological conditions.Recent studies have indicated the neuroprotective effect of VEGF and its protevtion effect in ALS has caused wide public concern.Many routes and vectors to delivery VEGF to CNS were applied.Azzouz et al.used the lentivirus as a vector to deliver VEGF through intramuscular injection and this method prolonged the lifetime of SOD1G93 A mice effectively.Other routes and vectors to delivery VEGF to CNS were applied,for instance,intracerebroventricular(ICV)injection of VEGF protein or adeno-associated virus 4(AAV-4)vectors for delivering VEGF.In another study,VEGFoverexpressing stem cells were transplanted into SOD1-G93 A mice via intrathecal injection.All these methods prolonged the lifetime of ALS transgenic mice effectively.However,we want to seek a more effective administration route and mode in order to improve transduction efficiency of VEGF.Studies have showed that AAV9 vectors with the cytomegalovirus(CMV)promoter show the highest gene expression in the motor neurons of the spinal cord.And although the intrathecal injection is invasive,it has the advantages of lower dose and less off-target effect.So,in our study here,we used an AAV9 vector with CMV promoter to transfer VEGF through intrathecal injection in order to improve its transduction efficiency,and then observed the effect of VEGF on the living phase of ALS mice.The mechanism of ALS is unclear;multiple complex mechanisms lead to motoneuron degeneration including the molecular and cellular mechanisms.Despite most intentions to the motoneuron injury,recent studies have revealed that the nonneuronal cells contribute to pathogenesis and disease progression.The non-cell-autonomous toxicity is crucial to motor neuron death.Microglial cells,a component of the innate immune system in the central nervous system(CNS),are key to accelerate the progression and play an important role in the inflammatory response.A recent study has showed that selectively inhibiting nuclear factor-kappa B(NF-kB),a major regulator of inflammation,in ALS microglias can rescue the motor neuron death.Microglias can be categorized into two different groups based on the stimulating factors and surrounding inviroment,the classically activated(M1)or alternatively activated(M2)phenotype.In early stage of the disease,the neuroprotective(M2)phenotype is predominant.However,during the rapid disease stage,there is a transition from a neuroprotective response to an injurious response resulting from production of several cytokines,such as tumor necrosis factor(TNF-?),interleukin(IL)-1 and interferon-?(IFN-?).So,based on the previous studies about the mechanism of VEGF,our study here explored the effect of VEGF on proliferation of microglia and neuroinflammation in ALS mice in order to further understand the neuroprotective effect of VEGF.Part one The effect of intrathecal injection of scAAV9-VEGF-165 on the survival and ethology of ALS miceObjective: To verify whether the improved intrathecal injection method could express the target genes effectively in CNS and to construct an AAV9-VEGF-165 vector with CMV promoter,then deliver the vectors to the female and male ALS mice of 90 days of age,and observe the lifespan and ethology of the ALS mice.Methods:1 We constructed the scAAV9-VEGF-165 and scAAV9-GFP vectors with CMV promoter,after analysis and purification;the vector titer was tested by PCR assay.2 We selected SOD1G93 A transgenic mice as animal model of the ALS,and the animals were breeding and reproduction in a constant temperature,constant humidity and sterile environment.The improved and simplified intrathecal injection method: Mice were first lightly anesthetized with chloral hydrate.A soft cloth was covered over the head and the upper body of the mice,and then gripped the mouse by the hip bones with the thumb and index finger and clipped hair of the waist in order to see the midline.Then a Hamilton needle was used to insert at the midline directly between the hip bones at a 70-80°angle to the horizontal,once the injectors sensed the bone of the spinal column the angle was reduced to about 30°and the experimenter attempted to slip in between the vertebrae(L5-6).A reflexive lateral flick of the tail or formation of an ‘S' shape by the tail could be a symbol of puncture of the dura.3 Three weeks after the intrathecal injection of scAAV9-GFP(1 x 109 vg/g),the mice were sacrificed and the tissues were removed freshly or after perfused with 4% icy paraformaldehyde.Then the expression and distribution of GFP were observed by immunofluoresence and immunoblot in order to confirm whether the target genes could express effectively in CNS by the improved intrathecal injection method.4 To intervene the female and male ALS mice of 90 days of age by the simplified intrathecal injection method.The littermate and bodyweightmatched female and male ALS mice of 90 days of age were divided into two groups randomly;the scAAV9-VEGF165 and scAAV9-GFP were giving according to the weight(1 ?l/g,n=15).We observed the lifespan and the bodyweight of the treatment and control groups,and assessed the motor function of the ALS mice by footprint analysis,rotarod and neurological score in order to observe the treatment effect of VEGF on ALS mice.Results:1 The vector titer of the constructed scAAV9-VEGF-165 and scAAV9-GFP vectors is 1 × 1012 vg/ml in PCR testing;2 Three weeks after the intrathecal injection of scAAV9-GFP,the immunofluoresence results showed that the GFP was expressed in the ventral horn of spinal cord mainly,and could be merged with the neurons labeled with NeuN.The immunoblot results showed that the GFP could be widely expressed in the CNS including spinal cord(mainly),cerebellum and cerebral cortex after the injection of scAAV9-GFP by an improved and simplified method;3 Compared with the control mice,the lifespan of the female VEGF-treated ALS mice was prolonged by 13 days(P?0.01)and the male mice was prolonged by 12 days(P?0.01),and there is no statistical differences between the female and male mice.In the early stage,the control group's body weight was lower than that of the VEGF-treated mice,but there is no statistical difference.Until the 121(female)and 129(male)days of age,the two groups displayed statistically significant difference.Footprint analysis showed a more obvious abnormality of gait and a shorter stride length in control group at 110 days of age.For rotarod tests,both the female and male treated mice performed better.Meanwhile,VEGF treatment could prevent the neurological scores decrease.Otherwise,haematoxylin and eosin staining of anterior tibial muscle section showed more serious muscle atrophy in GFP control mice and central migration of nuclei was also recognized in GFPtreated mice.Part two Intrathecal injection of VEGF protected the motor neurons by its anti-apoptotic effectsObjective: To observe the changes of the number and form of motor neurons in the spinal cord between the VEGF-treated and control ALS mice,and then study its mechanism of neuroprotection.Methods:1 We selected SOD1G93 A transgenic mice as animal model of the ALS,and the animals were breeding and reproduction in a constant temperature,constant humidity and sterile environment.At 90 days of age,6 groups of ALS mice were treated with scAAV9-VEGF-165 and scAAV9-GFP randomly,three weeks later,the mice were sacrificed and the tissues were removed freshly(3groups)or after perfused with 4% icy paraformaldehyde(3groups).2 To count the number of SMI-32 positive cells in VEGF-treated mice and the GFP control mice by immunohistochemical staing,and observe the form changes of the motor neurons between the two groups.3 To test the apoptosis in the spinal cord of the VEGF-treated mice and the GFP control mice,and test the changes of PI3K/Akt pathway along with the anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax.Results:1 The immunohistochemiscal staining results showed that the number of SMI-32 positive cells in VEGF-treated mice was higher than the GFP control mice,and the morphology was more intact(8.8 ± 1.96 vs.6.2 ± 1.3,P?0.05);2 The TUNEL results showed that the number of TUNEL positive cells was decreased obviously in the treatment group than the control group(24.8 ± 8.2% vs.44.5 ± 14.4%,P?0.05);3 Three weeks after the delivering of scAAV9-VEGF-165 by intrathecal injection,the level of VEGF-165 was increased to two-fold significantly.The immunoblot results showed that in the VEGF-treated mice the PI3K/Akt pathway was activated and the levels of the activated caspase-9 and caspase-3 were decreased(P<0.05).Meanwhile,the level of anti-apoptotic protein Bcl-2 was elevated,whereas the level of pro-apoptotic protein Bax was decreased in VEGF-treated mice(P<0.05).Part three VEGF administration could reduce microgliosis in the spinal cord of ALS mice and modulate microglia polarizationObjective: To observe the changes of the number and form of microglia and astrocytes in the spinal cord between the VEGF-treated and control ALS mice,and then study the effect of VEGF on microglia polarization and neuroinflammation.Methods:1 We selected SOD1G93 A transgenic mice as animal model of the ALS,and the animals were breeding and reproduction in a constant temperature,constant humidity and sterile environment.At 90 days of age,6 groups of ALS mice were treated with scAAV9-VEGF-165 and scAAV9-GFP randomly,three weeks later,the mice were sacrificed and the tissues were removed freshly(3groups)or after perfused with 4% icy paraformaldehyde(3groups).2 To count the number of GFAP positive cells and Iba1 positive cells in VEGF-treated mice and the GFP control mice by immunohistochemical staing,and observe the form changes of the microglia and astrocytes between the two groups.Moreover,we observed the difference of levels of CD11 b and GFAP between the VEGF-treated mice and the GFP control mice.3 We observed the expression of CD68 and its colocalization with Iba1 in the spinal cord of VEGF-treated mice,GFP control mice and the littermate,and then studied the changes of the mRNA levels of CD68,TNF-?,IL-1?,Arginase-1 and Ym-1in the spinal cord of VEGF-treated mice,GFP control mice and the littermate.The immunoblot was used to test the changes of CD68,TNF-?,Arginase-1and TGF-? in the spinal cord of VEGF-treated mice,GFP control mice and the littermate.Results:1 The immunohistochemiscal staining results showed that the number of GFAP positive cells(311 ± 46 vs.163 ± 17.61,P<0.05)and Iba1 positive cells(383 ± 19.15 vs.218.3 ± 28.02,P<0.055)in GFP control mice was higher than the VEGF-treated mice,and the morphology was more enlargements.Consistent with the immunohistochemiscal staining results,the immunobolt results showed that the levels of CD11 b and GFAP were decreased in the VEGF-treated mice;2 The immunofluoresence results indicated that there is a few of CD68 positive cells in the spinal cord of the littermate mice,and the number of CD68 positive cells was increased obviously in the GFP control ALS mice,and this could be inhibited by the VEGF treatmen.Meanwhile,the CD68 positive cells could be merged with the microglia labeled by Iba1;3 The PCR results showed that the markers of M1microglia(CD68,TNF-?,IL-1?)was decreased in VEGF-treated mice(P<0.05),however the markers of M2 microglia(Arginase-1and Ym-1)was elevated(P<0.05).4 Consistent with the PCR results,the immunobolt results showed that VEGF treatment could increase the level of Arginase-1 and decrease the expressions of CD68 and TNF-?.Meanwhile,the level of TGF-?,which could promote the microglia into the M2 type,was also increased after the VEGF treatment.Conclusions:1 In our study here,we simplified and improved the intrathecal injection method,and we constructed an AAV9 vector with CMV promoter to transfer target genes.After delivering the scAAV9-GFP,we found that the target gene could be widely expressed in CNS,and were expressed mainly in the motor neurons in the ventral horn of spinal cord.Based on our results,the feasibility of this method was confirmed,and laid a foundation for the treatment of VEGF.2 Consistant with the previous study,the lifespan of female and male ALS mice was prolonged by scAAV9-VEGF through our method,and the motor function was obviously improved.Our results further confirmed the therapeutic effect of VEGF in ALS.3 The motor neurons of ALS mice were preserved after the treatment of VEGF.We further confirmed that the VEGF's neuroprotective role was benefited from the activation of the PI3K/Akt pathway.And the level of pro-apoptotic protein was decreased,whereas the anti-apoptotic protein level was increased.4 Microgliosis plays an important role in the progression of the disease,and we found that VEGF administration could reduce microgliosis effectively and modulate microglia polarization. |
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