Intraspinal injections - To Laminectomy or Not?

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Intraspinal viral injections are increasingly used to study spinal circuits. Traditionally, the approach would involve the removal of the vertebrae overlying the region of interest (usually L3-L5), providing an open territory for injection.

Here is an exemplar of that approach: https://www.jove.com/video/50313/stereotaxic-injection-viral-vector-for-conditional-gene-manipulation

Recently, Kohro et al. reported a method that circumvents laminectomy and accesses the spinal cord through the interspace between the spinous processes of T13 and L1.

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https://www.nature.com/articles/srep14306

Each method has its advantages. The full laminectomy version allows greater territory to be injected, with multiple injections (e.g. Foster et al 2015), but it also is very invasive and I fear that the area of exposed spinal cord is prone to infection and scarring.

The Kohro method avoids the damage of the laminectomy but really only allows a single injection, which could limit the spread of the virus to smaller area (L4 in their paper). Peirs et al (@rpseal lab) used an approach on very P9-P21 animals using the non-laminectomy approach to good effect.

I’ve tried both approaches this summer here at the MBL, and I’ve settled on the non-laminectomy method for now. It’s actually not that hard. I thought it was going to be challenging to find that space, but with a little tweezing and dissection, and the right stereotactic setup (Nashige spinal one here), it’s not hard. More challenging is filling the glass pipette properly with mineral oil and not clogging the tip, and also breaking through the dura. If anyone has tips on that, please share.

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Clamping the cord and finding the interspace is not that hard

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Dye injection demonstrating proper location
What are your thoughts and experiences? Which do you prefer and why? With the non-laminectomy method, would you do another injection, say at the T12/T13 interspace or would this be too high? It looks like it could be L2-L3, depending on strain.

@zhzhj131421 @zhisrich @liz @fmoehring @tberta @sshiers @SamineniV @runDRG

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I don’t have experience doing spinal cord injections, but the possibility of injection without laminectomy is very exciting! I’ve always been concerned with the effect of the laminectomy itself–in terms of causing additional pain and impacting the biomechanics of the spine. It’s not quite the same, but in orthopaedics, we have groups modeling arthritic pain in the spine by damaging the facets of the spinous processes, which both induces pain and significantly changes the way the spine is loaded, so I would not be surprised to see somewhat similar effects (although likely on a smaller scale) with laminectomy. So to have a model without that potential effect is pretty neat. For trying to go more rostral with this method, it may be difficult, as the ribs may impair your ability to manipulate the spine in the way necessary for exposing the cord. Have you seen anyone try this in larger models, such as rats or rabbits?

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@runDRG Thank you for your insights. Avoiding bone damage is why I prefer the non-lami method. I can definitely see the interspace between T12 and T13. I think I could do it. I’ll let you know.

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Hi Alex,

Thanks for the sharing! I think you must be very good at intraspinal injection now. I am currently also working on this technique. I found it is almost impossible for me to avoid the motion of the spinal cord even though I have clamped the vertebral column very tight. I think the motion of the spinal cord is due to the breathing. I can see the dye come out from the injection site during infusion (I used a minipump and injection rate is about 30 nl per min, and the depth into spinal dorsal horn is 300 micrometers). Can you please share your experience of avoiding this motion and leakage? Thanks a lot!

Hongsheng

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Hi @whshhaibing123

I haven’t done this in a while actually, but are you talking about motion in the anterior-posterior axis or left-right or rostral-caudal? With breathing, it’s inevitable there will be a little rostral caudal. Are you getting any dye injection? How do you know it’s not going in?

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Appreciate the quick response, Alex! You are right, I saw the rostral caudal motion. I tested with dye and confirmed they got in by cutting slices and check. What I am worrying about is how much they can get in when I do the virus injection. I just started and haven’t check the virus yet. Based on your experience, how much volume is necessary for AAV to diffuse through the whole dorsal horn in the injected region? Maybe the leakage is not a big issue if we increase the volume of AAV?

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People generally use 500-1000 nl.

Here is an example from Peirs et al 2015

SNI was preformed as previously described. Spinal cord injections of AAV were performed at P9–P10 or P15–P16. Under isoflurane, midline incision was made without laminectomy and virus delivered (1 ul) slowly with a glass microelectrode (50 mm tip) between lumbar segments L4 and L5. Silk sutures were used to close lassimus dorsi, and skin and Ketofen was given before and 1 day after surgery. Behavior was tested 3 weeks later. For more details including virus titer, see Supplemental Experimental Procedures. @RPSeal

Similarly, this JoVe video from Inquimbert et al. uses 1 ul

If you can see the dye, it’s getting in. As far as titer goes, people are usually using 10^12 to 10^13 range. You will need to test yourself.

Here are some more resources:

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Appreciate so much! I’m going to test and share my results!

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