TITLE: Transplantation of Fetal Midbrain Dopamine Progenitors  into a Rodent Model of Parkinson’s Disease
AUTHORS: Lachlan H. Thompson, Clare L. Parish

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Cell therapy is a promising experimental treatment for Parkinson’s disease (PD). It is based on the idea that new dopamine neurons transplanted directly into the forebrain of the patient can structurally and functionally compensate for those lost to the disease in order to restore motor function. While there is a highly active field of research focused on the development of stem cell-based procedures, fetal tissue remains the “gold standard” as a safe and reliable source of dopamine neuron progenitors capable of structural and functional integration with existing motor circuitry following transplantation. This chapter describes the basic procedures for preparation of dopamine progenitor rich cell suspensions of ventral mesencephalon as well as implantation into the unilateral 6-hydroxydopamine model of PD and assessment of functional impact according to drug-induced rotational behavior. The description assumes a basic knowledge of animal handling and stereotaxic surgical procedures in rodents.</div></div>

[STEPS]
?. [3.1 Preparation  of Cell Suspensions]
Remove the uterus immediately from euthanized or terminally anesthetized animals via caesarian section, place in cold L15 medium (see Note 2) and store .
on ice
?. [3.1 Preparation  of Cell Suspensions]
Place the embryos in a petri dish containing ice-cold L15  medium. Under a dissection microscope (see Note 1), remove  each embryo using suitably fine dissection instruments. This is best achieved through a single incision parallel to the placental mass (see Fig. 1a, b) while applying gentle pressure with the forceps to both sides of the sac—the positive pressure in the  amniotic sac will usually expel the embryo. Cut the umbilical cord (Fig. 1c) and repeat for the next embryo.
?. [3.1 Preparation  of Cell Suspensions]
The ventral mesencephalon can be obtained from each embryo in five steps (step 4 - step 8) using forceps to stabilize the embryo and scissors to isolate the VM as a single piece of tissue (Fig. 1):
?. [3.1 Preparation  of Cell Suspensions]
(a) The anterior end of the mesencephalic tube is cut at approximately the di-mesencephalic boundary—parallel and directly adjacent to where the narrow tube expands into the larger forebrain regions containing the developing cortex and thalamus (Fig. 1e).
?. [3.1 Preparation  of Cell Suspensions]
(b) The caudal end of the mesencephalic tube is cut at approximately the level of the mid-hindbrain boundary (Fig. 1e;  see Note 3).
?. [3.1 Preparation  of Cell Suspensions]
(c) The mesencephalic tube can now be isolated by sliding one blade of the scissors between the meningeal layers and  the VM—perpendicular to the cuts performed in step 4 and step 5  (a, b)—in order to cut the connective tissue overlaying the  midbrain (Fig. 1f, g).
?. [3.1 Preparation  of Cell Suspensions]
(d) The ventral midline is exposed by “rolling” the VM tube  90° away from the user (Fig. 1h). The dorsal midline will  remain attached to the meningeal tissue, face-down on the petri dish. Cut parallel to the midline approximately 1/3  of the distance between the ventral and dorsal midline on one side of the VM tube (Fig. 1h).
?. [3.1 Preparation  of Cell Suspensions]
(e) Perform a second cut at the same position on the other side of the tube to free the final VM piece used for transplantation (Fig. 1h; see Note 4). It may be necessary to  trim excess tissue and it is important to peel away any  remaining meningeal tissue (see Note 5).
?. [3.1 Preparation  of Cell Suspensions]
Remove the media from the eppendorf containing the pooled VM pieces and replace with medium that does not contain Ca2+ or Mg2+ (e.g., DMEM/PBS/HBSS-Ca 2+/-Mg2+) and is supplemented with  and . Incubate at   for  (see Note 6).
[DNase]
[trypsin]
37 °C
?. [3.1 Preparation  of Cell Suspensions]
Rinse the VM pieces in medium-Ca2+/-Mg2+ three times before adding a known volume of medium-Ca2+/-Mg2+ with  for tissue dissociation (see Note 7).
[DNase]
?. [3.1 Preparation  of Cell Suspensions]
Mechanically dissociate the VM tissue pieces by gently trituration with a 1 ml pipette tip followed by a 200 µl tip. Continue as necessary to achieve a cloudy suspension.
?. [3.1 Preparation  of Cell Suspensions]
To estimate the total viable cell number, dilute a small (e.g., –) sample of the suspension 1:10 in Trypan blue and count viable (dye-excluding) cells using a hemocytometer. Total viable cells = cells counted × dilution factors (e.g., trypan dilution and fractional area of hemocytometer counted) × volume of suspension. Cell viability should be >90%.
1 µl
5 µl
?. [3.1 Preparation  of Cell Suspensions]
Centrifuge at . Remove the supernatant and use a pipette to resuspend the pellet in medium-Ca2+/- Mg2+ with  at the desired cell density (see Note 8). Be careful not to introduce air bubbles when resuspending. Store  throughout transplantation procedure.
Centrifuge: 500 34, 5 min, 4 10
[DNase]
on ice
?. [3.2 Unilateral Lesioning of the Nigrostriatal Dopamine System in Rodents]
Prepare the 6-hydroxydopamine (6OHDA) solution by dissolving 6OHDA in sterile saline () containing  to achieve the desired concentration (see Note 9).
[L-ascorbic acid (Sigma)]
Do not use PBS, this will oxidize the toxin (see Note 9).
?. [3.2 Unilateral Lesioning of the Nigrostriatal Dopamine System in Rodents]
Rodents should be deeply anesthetized with the head placed in a fixed-skull position using a stereotaxic frame.
?. [3.2 Unilateral Lesioning of the Nigrostriatal Dopamine System in Rodents]
Make an incision in the scalp along the midline and drill a small burr hole in the skull at the desired coordinates (see Note 10) to reveal the dural surface.
?. [3.2 Unilateral Lesioning of the Nigrostriatal Dopamine System in Rodents]
Inject the 6OHDA solution using a micro-volume syringe, preferably fitted with a pulled glass cannula (see Note 11).  Leave the cannula in place for at least  after injection to allow for diffusion of the toxin. If necessary, the central noradrenergic system can be protected by intraperitoneal injection of desipramine (; Sigma) 30 min before injection of  6OHDA (see Note 12).
?. [3.2 Unilateral Lesioning of the Nigrostriatal Dopamine System in Rodents]
Close the scalp (e.g., using suture or Michel clips). A topical anesthetic (e.g., lidocaine) and/or a central analgesic (e.g., Temgesic 0.3 mg/kg) may be administered. This is particularly relevant for short recovery times, e.g., when using inhaled anesthesia.The functional impact of unilateral lesioning of the nigrostriatal pathway can be readily determined by measuring turning behavior in response to D-amphetamine or apomorphin.
?. [3.2 Unilateral Lesioning of the Nigrostriatal Dopamine System in Rodents]
For D-amphetamine-induced rotation, dissolve D-amphetamine in  and administer (i.p.)  for rats or  for mice. For apomorphine-induced rotation, dissolve apomorphine in  with  administer (s.c.) for rats or  for mice (see Note 13).
[sterile saline]
[saline]
[L-ascorbic acid]
?. [3.2 Unilateral Lesioning of the Nigrostriatal Dopamine System in Rodents]
Allow the animals to habituate for  after drug injection. Record the number of full (360°) body rotations in each direction over a defined time period—typically – for apomorphine and – for amphetamine (see Note 14).
?. [3.2 Unilateral Lesioning of the Nigrostriatal Dopamine System in Rodents]
Record the rotation score for each animal as net full body rotations per minute in one direction (see Note 15).
?. [3.3 Implantation of Cell Suspension]
Prepare deeply anesthetized animals in a sterotaxic frame and drill a burr hole through the skull at the desired coordinates as per steps 15 and 16 (see Note 16).
?. [3.3 Implantation of Cell Suspension]
Resuspend the cells through gentle titruation using a pipette to achieve a uniform suspension.
It is important to repeat this step prior to each implantation as an effort to deliver a consistent number of cells across all animals.
?. [3.3 Implantation of Cell Suspension]
Prefill the microsyringe and glass cannula with vehicle (e.g.,  or cell medium) and draw the required volume of cells into the glass cannula through the tip (unlike when using aqueous solutions, there is no need to draw an air-bubble first).
[saline]
?. [3.3 Implantation of Cell Suspension]
Slowly pressure-inject the cells at the desired location. It is important to monitor cell-flow through the glass cannula  throughout the injection to ensure that all the suspension has been injected (this may not be the case if there is a blockage or a leak in the cannula setup) and, importantly, that there is no backflow of the injected contents during the injection (see Note 17). Keep the cannula in place for at least  before slowly withdrawing.
?. [3.3 Implantation of Cell Suspension]
Close the scalp and administer local anesthetic and central analgesia as per step 18 above.
?. [3.1 Preparation  of Cell Suspensions]
Rinse the VM pieces in medium-Ca2+/-Mg2+.
?. [3.1 Preparation  of Cell Suspensions]
Rinse the VM pieces in medium-Ca2+/-Mg2+.
?. [3.1 Preparation  of Cell Suspensions]
Rinse the VM pieces in medium-Ca2+/-Mg2+.
?. [3.1 Preparation  of Cell Suspensions]
Add a known volume of medium-Ca2+/-Mg2+ with  for tissue dissociation.
[DNase]