Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.
After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16 °C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 °C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.
In mammals spinal cord injury (SCI) is a devastating condition that leads to permanent loss of function below the site of injury because injured axons do not regenerate through the trauma zone and reconnect to their appropriate targets. In contrast to mammals, lampreys recover locomotion after a complete spinal cord injury.1 Interestingly, lampreys have a set of 36 spinal cord projecting neurons that are individually identifiable in whole-mount brain preparations because of their big size2,3 (Figure 1). All of these spinal-projecting neurons are axotomized by a high-level complete spinal cord transection. Previous studies of our group and others have shown that even in the presence of functional recovery after SCI some of these neurons show a very low regenerative capacity (they are considered “bad regenerators”), while others usually regenerate their axon through the site of injury (they are considered “good regenerators”).2,3 This characteristic makes lampreys an interesting vertebrate model to study the differences in gene expression between good and bad regenerator spinal-projecting neurons that in turn will lead to the differences in the intrinsic regenerative ability of neurons that attempt to regenerate their axons in the same extrinsic environment.1
Using this model we have previously shown that spinal-projecting neurons with low regenerative ability show expression of axonal guidance molecule receptors like UNC54,5 and neogenin,6 which mediate the inhibitory action of netrin and RGM respectively. In addition, by using this method our group has also shown that only the good regenerators show a recovery of the expression of neurofilaments after the injury and during the regeneration process. Recently, Busch and Morgan7 have shown by immunofluorescence that the bad regenerators show an increased expression of synuclein after injury, which has been related by the authors to the fact that the “bad regenerator” spinal-projecting neurons slowly die after a complete spinal cord transection5,7,8. So, the lamprey model of a complete spinal cord injury has emerged as a very useful model to understand what makes a spinal cord projecting neuron a “bad regenerator” after axotomy.
To conduct our studies we are performing a complete spinal cord transection surgery protocol and a posterior brain dissection at the desired time points after injury to perform wholemount in situ hybridization. In the present methodological article we present a detailed protocol for the proper performance of a complete spinal cord injury surgery in larval lampreys, the subsequent maintenance of the animals and the final brain dissection and preparation of the brain for a wholemount in situ hybridization. A detailed protocol to perform the wholemount in situ hybridization in the brain of larval lampreys has been previously reported.9 In addition, this protocol for spinal cord injury and brain dissection can be also used to then process the brains for immunohistochemistry or other histological methods.
See Table 1 for all materials used in this protocol.
Experiments were approved by the Institutional Animal Care and Use Committee at Temple University.
1. Animals
2. Complete Spinal Cord Transection
3. Brain Dissection and Preparation of the Brain for in situ Hybridization
As an example of the results that can be obtained when using this method, representative images of wholemounted brains showing the expression of the neogenin transcripts in identifiable spinal cord-projecting neurons of control and 2 weeks post lesion larval sea lampreys are shown in Figure 2. The readers are referred to a previous study6 reporting the relationship between the expression of neogenin after a complete spinal cord transection and the regenerative ability of the identifiable spinal cord projecting neurons of the sea lamprey for full details. Briefly, results have shown that after the injury there are changes in the expression of the neogenin receptor in identifiable spinal-projecting neurons. Two weeks after a complete spinal cord transection the neogenin receptor is preferentially expressed in bad regenerators, like the M1, I1, I2, I4, or Mth neurons (see Figure 2B) These results demonstrate the outcome of the protocol and suggest that the neogenin receptor could be involved in the failure of the regeneration of these cells.
Figure 1: Schematic drawing of a dorsal view of the sea lamprey brain showing the location of identifiable reticulospinal neurons. Reproduced from Barreiro-Iglesias and Shifman.5 For abbreviations, see list below.
Figure 2. Photomicrographs of dorsal views of the mature larval sea lamprey brain showing the expression of the neogenin transcript in control (A) and 2 weeks post-injury (B) animals. Note in B that after the injury the neogenin transcript is preferentially expressed in neurons known to be bad regenerators (e.g., M1, I1, I2, I4 and Mth neurons). Also, note the presence of the holes left by the pins used to hold the brain (arrows). The readers are referred to the previous study by Shifman and coworkers.6 For abbreviations, see list below.
ABBREVIATIONS
B (B1-B6) | Müller cells of the bulbar region (middle rhombencephalic reticular nucleus) |
hab.-ped. tr. | habenulopeduncular tract |
I1-I6 | Müller cells of the isthmic region |
IX | glossopharyngeal motor nucleus |
inf. | infundibulum |
isth. retic. | isthmic reticular formation |
M1-M4 | Müller cells 1 to 4 |
Mth | Mauthner cell |
mth’ | auxiliary Mauthner cell |
s.m.i. | sulcus medianus inferior |
Vm | trigeminal motor nucleus |
X | vagal motor nucleus |
Here we present a detailed protocol to perform a complete spinal cord transection and posterior brain dissection in larval sea lampreys. This procedure allows analyzing differences in gene expression between identifiable spinal cord projecting neurons after spinal cord injury by means of a whole-mount brain in situ hybridization. The critical step in the procedure is the correct performance of a complete spinal cord transection, which can be controlled by observing the cut ends of the spinal cord under the stereomicrocope and confirmed 24 hr later by gently touching the snout of the larva with a pair of forceps to see if there is no movement caudal to the site of injury. It is also important for the recovery of the animal that the scissors used for the surgery are not blunt and that a single and clean cut is done to completely transect the spinal cord.
The survival of the animals after the injury is highly improved by keeping the animals on ice during the surgery and during the hour used to allow the wound to air dry and then in ice cold water for the first 24 hr. After this period the wound is closed and the animals can already be transferred to tanks with freshwater at room temperature.
Larval lampreys are allowed to recover at room temperature and not at their usual cold temperature because a greater majority of the animals recover full locomotor behavioral function after the injury at higher temperatures.11 The recovery of a normal pattern of locomotion is observed 8 to 10 weeks after the complete spinal cord transection.
After performing the complete spinal cord transection and the brain dissection at the chosen time points the larval brains can be processed not only to perform a whole-mount in situ hybridization as shown here (Figure 2), but also for other histological methods like immunohistochemistry (e.g. 7), the detection of neuronal tracers that can be applied at the time of transection at the spinal cord injury site (e.g. 8) or the detection of activated caspases by using fluorochrome labeled caspase inhibitors.5
The authors have nothing to disclose.
Supported by NIH Grants NS14837, R01 NS38537, R24 HD050838 to Dr. Michael E. Selzer; Shriners Research Grant SHC-85220 to Dr. Michael E Selzer; and Shriners Research Grant SHC-85310 to Dr. Michael I. Shifman. Dr. Antón Barreiro-Iglesias was supported by the Fundación Barrié (Spain) and the Xunta de Galicia (Galicia, Spain).
Name of the reagent | Company | Catalogue number | Comments (optional) |
Tricaine methane sulfonate | Spectrum | TR108 | Benzocaine saturated solution in PBS for sacrifice |
Scalpel #3 | Fine Science Tools (FST) | 10003-12 | |
Blades for scalpel: #11 | Fine Science Tools | 10011-00 | |
Castroviejo scissors #8 | Fine Science Tools | 15002-08 | |
Forceps #4 & #5 | Dumont, Switzerland | Roboz RS4955 | #4 for dissection of Spinal cord; #5 for stripping menninges |
Dissecting Microscope | Olympus | SZ51 | |
Sylgard | Dow Corning Co. | 184 | |
Insect pins 0.15, 0.20 mm | Austerlitz | No catalogue # | 0.15 mm for pinning brain and spinal cord; 0.20 mm for the body |
7 ml HDPE Scintillation Tubes with Caps | Fisher Scientific | 03-337-1 | |
Paraformaldehyde 16% | Electron Microscopy Science (EMS) | 19210 | Dilute to 4% in PBS |