Anatomy 101

Sometimes there are technological advances that truly take us to a new playing field, and we’ve been lucky to be part of one this year. It’s going to take a bit to describe the technology, so find a comfortable chair and settle in. But before getting into the weeds of the technology, let’s set the stage in terms of what we need to repair the injured spinal cord.

We scientists tend to focus on paralysis because it’s what is most obvious after spinal cord injury (SCI). Also, loss of ability to move and movement recovery are something we can measure and quantify in experimental animals, so these are usually the main “outcome measure” we scientists use for our spinal cord injury research programs. Our group’s focus has been on the corticospinal tract (CST) which controls our ability to move voluntarily, and even short distance regeneration of CST axons could have a huge impact on quality of life for people whose hands and arms are paralyzed as a result of a cervical spinal cord injury (the most common type of injury in people).

But all of you living with spinal cord injuries know that paralysis is only part of the story. Loss of ability to feel below the injury (loss of sensation) is important, and of course loss of bladder, bowel, sexual function and autonomic regulation of temperature and blood pressure are major problems that can be life threatening. All of these functions are mediated by multiple different pathways from the brain to the spinal cord, and the greatest hope for recovery of multiple functions is to regenerate multiple pathways that are interrupted by SCI.

Now, on to the science:

Scientists at RIRC and throughout the world have been working diligently to find ways to stimulate regeneration of connections with some recent success. For example, our focus has been on using AAV vectors carrying shRNA to knock down the gene PTEN in cortical neurons to boost intrinsic growth capacity and thus stimulate regeneration of CST axons. We do this by injecting AAV/shPTEN into the cerebral motor cortex (seeAnatomy 101, Fall 2018 issue of Spinal Connections). The limitation, however, is that we need to make multiple small injections to target a sufficient number of cortical neurons, and even then, we don’t get to the neurons of other spinal pathways at all. So, the best we can hope for is regeneration of some CST axons, which is important, but only part of the answer.

The huge breakthrough that we’ve achieved over the past year is that we’ve been able to build on major discoveries in viral vector biology to develop a new type of AAV to deliver gene modifying cargoes to neurons whose axons are damaged by SCI. This is called “retro-AAV”, which has the remarkable property that it can be injected into the spinal cord, where it’s taken up by axons and transported back to the cells of origin of spinal pathways in the brain.

This is called retrograde axonal transport (the reason that the new AAV is called retro-AAV). Now imagine—instead of making injections of AAV into the brain, we can make a single injection into the spinal cord and the AAV with its cargo is transported back to neurons in the brain that give rise to multiple pathways that are damaged by spinal cord injury.

A picture is worth a thousand words, so here’s the picture (Figure). In this study, we used a retro-AAV that expresses the enzyme Cre recombinase (retro-AAV/Cre) in our transgenic strains of mice. These mice are derived from the mice we used for our studies of regeneration enhancement with PTEN deletion. The mice are genetically modified so that when AAV/Cre transfects neurons, expression of Cre triggers a rearrangement of the mouse’s DNA to delete PTEN and turn on expression of a fluorescent reporter protein called tdTomato.

Panel A of the figure shows an intact brain and spinal cord from a mouse with AAV/Cre injection at cervical level 5 (the bright spot in the spinal cord indicated by “inj”). Amazingly, you can see the glow from thousands of red fluorescent neurons that give rise to the CST (CMNs) in the intact brain. Panel B illustrates this brain using light sheet microscopy—another new technology we’re deploying. For this, the brain is placed in chemicals that make it transparent, so now you can make out the milky way of light spots indicating individual neurons. There are about 25,000 transfected neurons in this milky way (about 80% of CST neurons are transfected). The individual CMNs are seen more clearly in slices through the brain (panel C and D).

The key thing is that neurons of origin of other spinal pathways are also transfected (shown in panels E-Gin green fluorescence to highlight the fact that these neurons mediate other functions). For example, neurons in the red nuclei (E) contribute to voluntary motor function; neurons in the reticular formation (F) control walking and posture; and maybe best of all, neurons in Barrington’s nucleus (BN in G) control bladder function.

So, now we are set to test whether it’s possible to use the retro-AAV technology to trigger regeneration of multiple pathways after SCI to restore multiple functions including motor control of the hands and arms, walking, and yes, even bladder. Os Steward and his research team were awarded a new $1.5 million NIH grant in April, 2019 to further develop the retro-AAV technology for spinal cord injury therapy.

If a picture is worth a thousand words, a movie is worth a thousand pictures, right? Actually, the image in panel B IS made up of over a thousand transparent pictures of slices through the intact brain taken with the light sheet microscope. The slices are then assembled into a 3D reconstruction (similar to an MRI) that you can rotate and see from different angles. If you want to see the movies of these reconstructions, links are on our website (Reeve.uci.edu).

We’re really excited about this breakthrough and look forward to telling you more in future issues of Spinal Connections.

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