The overall goal of this procedure, is to reproduce most of the essential aspects of Parkinson's disease in an animal model. The stereotaxic delivery of adeno-associated viral vectors, encoding the human synuclein in the substantia nigra. Parkinson's disease is a neurodegenerative disorder that involves the death of dopaminergic neuron, in the nigrostriatal pathway, and consequently, the progression of loss of control of voluntary movement.
This neurodegenerative process is accompanied, by deposition of protein aggregate in the brain, which are mainly constituted by synuclein. Emerging evidence has indicated, synuclein aggregates can stimulate toll-like receptors on the microglia, thus triggering neural inflammation in the substantia nigra. Furthermore, evidence indicate that synuclein aggregates can be captured and presented, by antigen-presenting cells to T cells, inducing an adaptive immune response specific to synuclein.
The stereotaxic delivery of adeno-associated viral vectors encoding the human synuclein in the substantia nigra, has shown to reproduce many essential aspects of the disease, including synuclein pathology, microglial activation, T-cell activation, neurodegeneration, and motor impairment. This study presents an analysis of how a viral vector and the time after viral vector delivery, affects the extent on human synuclein expression, neurodegeneration, and neuroinflammation in the nigrostriatal pathway, as well as the degree of motor impairment in a mouse model of unilateral stereotaxic delivery of human synuclein in substantia nigra. To maintain an aseptic environment, wear appropriate surgical clothes during the whole surgery, including shoe covers, surgery mask, sanitary barrier, gloves, and surgery cap.
Spray ethanol on the mouse and all surgical material, to maintain the aseptic environment. To induce an analgesia, inject carprofen subcutaneously every 12 hours, starting one hour before the surgery, and continuing until three days after the surgery. To anesthetize the mouse, place the animal in an induction chamber.
Open the isoflurane flow at a rate of 0.5%and then slowly increase it up to 5%over approximately five minutes until the mouse has lost its righting reflex. Remove the animal from the induction chamber. Immediately transfer the animal to a non-rebreathing circuit with an appropriately-sized nose cone.
Maintain the mouse anesthesia, with isoflurane 1%throughout the whole time of surgery. Confirm the mouse is entirely anesthetized by pinching its tails and paws. When the mouse doesn't react when pinching the tail and paws, it means the mouse is completely anesthetized.
Shave the mouse head using scissors. Clean the mouse skin using a cotton swab with chlorhexidine 2%and remove all the hair. Fix the mouse head in the stereotaxic frame.
Place a corneal protectant in both mouse eyes, using a cotton swab. To prevent stress induction in other rodents, avoid the presence of any other mouse in the surgery room. Clean the mouse head with three rounds of chlorhexidine 2%followed by ethanol 70%Expose the skull using surgical material, and make a thin hole with a drill at coordinates anteroposterior 2.8 mm, and mediolateral 1.4 mm, with respect to medial line.
Put the needle of a 10 microliter syringe in the hole, and move the needle inside the brain slowly, until arriving to 7.2 mm dorsoventral, respect to dura. Leave the needle in the final position for two minutes, to allow the tissue to settle a bit, and then inject one microliter of viral vectors into the right substantia nigra, at a rate of 0.2 microliters every 30 seconds. Leave the needle in the same position for five minutes after the delivery of viral vectors, and then withdraw it slowly.
Close the wound using a sterile silk suture. Put the mouse in the home cage, pre-warmed by placing it over an electric warm mattress. 12 weeks after the stereotaxic surgery, assess the motor performance, using a simplified version of the beam test, described before.
For this purpose, use a horizontal beam of 25 cm in length and 3 cm in width. The beam surface must be covered by a metallic grid with squares of one centimeter, and elevated one centimeter above the beam. Make a video of the mouse, while traversing the grid surface beam, from one end to the opposite end of the beam, where the home cage is located.
Train the mouse for two days, before the determination of the motor performance. On the first day, train the mouse to walk through the beam five times, without the grid. On the second day, train the mouse to walk through the beam, in the presence of grid five times.
On the third day, evaluate the motor performance. To do this, quantify the number of errors carried out, by the left paws or by the right paws separately, by watching the videos in a slow-motion mode. To anesthetize the mouse, inject a mixture of ketamine and xylazine intraperitoneally.
Once the mouse is completely anesthetized, open the thorax with surgical material, and expose the heart. Then insert a flat tip needle, into the heart's left ventricle. By coupling the needle to a pipe, perfuse 50 milliliters of phosphate buffer saline, at a rate of 9.5 milliliters per minute, using a peristaltic pump.
Remove the brain using scissors and tweezers, and then fix it by immersion, in five milliliters of 4%paraformaldehyde, in phosphate buffer saline. Afterward, put the fixed brain, in 15 milliliters of 30%sucrose for 48 hours. Then put the brain in four milliliters of cryoprotection solution, and save the brain at 80 degrees, or use it immediately in the next step.
To obtain striatal slices, cut the brain into 40 micrometers thick sections, starting at 1.34 mm, and finishing at 0.26 mm. Harvest each slice in a two milliliters cryotube, following anteroposterior order. To carry out immunohistochemical and immunofluorescent analysis in the striatum, choose five coronal striatal sections taken at uniform intervals, that cover the entire rostrocaudal extent of the nucleus.
To validate the correct delivery of viral vectors in the dopaminergic neurons of the nigrostriatal pathway, vectors encoding GFP were injected, in the substantia nigra, and 12 weeks later, the GFP fluorescence and tyrosine hydroxylase immunoreactivity were analyzed in the substantia nigra and striatum by immunofluorescence. The GFP-associated fluorescence, was located exclusively in the ipsilateral site, and a significant colocalization with tyrosine hydroxylase immunoreactivity was observed in both substantia nigra and striatum, indicating the correct delivery of viral vectors in the dopaminergic neurons of the nigrostriatal pathway. To test the dose of vectors, encoding synuclein required, to induce a significant overexpression of human synuclein that promotes neurodegeneration of the nigral dopaminergic neurons, different doses of the vectors were injected.
And 12 weeks later, the human synuclein immunoreactivity, and the extent of tyrosine hydroxylase immunoreactivity were tested in the nigrostriatal pathway. Human synuclein immunoreactivity was evident, with all doses tested in the substantia nigra. However, only mice receiving 10 to the 10 viral genomes per mouse, presented evident human synuclein immunoreactivity, in the striatum.
Mice receiving 10 to the 10 viral genomes per mouse of vectors encoding human synuclein, displayed a significant loss of dopaminergic neurons in the substantia nigra. Although mice receiving the same dose of vectors encoding GFP, displayed a low degree of neuronal loss, these mice presented significantly lower degree of neurodegeneration, in comparison with those mice receiving vectors, encoding human synuclein. Using the beam test, a significant reduction in motor performance was detected exclusively in those mice, receiving 10 to 10 viral genomes per mouse of vectors encoding human synuclein, when comparing the number of errors made by the right and the left paws, or when comparing the total number of errors, of mice receiving vectors encoding human synuclein, with those mice receiving the control vector.
To define the onset of human synuclein expression, mice were treat with 10 to the 10 viral genomes per mouse, of vectors encoding human synuclein, or sham surgery, and the extent of human synuclein expression was analyzed in the substantia nigra, once a week, during weeks two to five after the stereotaxic surgery. The results show that human synuclein expression started being evident at week five after the stereotaxic surgery. To determine the peak of microglial activation, the extent of cells expressing high levels of Iba1, was quantified in the striatum of mice, during weeks 2-15 after the surgery.
The results show a significant increase in microglial activation of the ipsilateral side, compared with the contralateral side of mice, 15 weeks after the viral inoculation. The number of regulatory T cells infiltrated into the substantial nigra was analyzed at different time points, after the stereotaxic surgery by immunofluorescence, followed by confocal microscopy observation. The peak of regulatory T cell infiltration into the substantia nigra was observed at week 11 after surgery.
The mouse model of neurodegeneration analyzed here, represent a useful model to study numbers of key aspect involvement in pathophysiology of Parkinson disease. Involvement mechanisms involved in synuclein pathology, microglial activation, the involvement of the peripheral immune system, in neuroinflammation, and the mechanisms of neurodegeneration A proper dose of adeno-associated viral vector, subtype five encoding human synuclein to induce neurodegeneration, neuroinflammation, T-cells infiltration, and motor impairment, is 10 to the 10 viral genomes per mouse. This study shows, that five weeks after the stereotaxic surgery, constitutes a key time point, in which human synuclein expression was already evident, but in the absence of motor impairment in this animal model.
Thereby, this time point, represents an interesting temporal point, to start the administration of experimental therapies tailored to stop the progress of neuroinflammation and neurodegeneration. The most suitable time point to analyze neuroinflammation in this animal model, is at week 15 after a stereotaxic surgery. While a proper time point to analyze T-cell infiltration into the central nervous system.
seems to be at week 11 after viral vector inoculation.
