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At the Bench-Stroke Recovery
Researchers Uncover Molecular Signature for Recovery from Stroke

ARTICLE IN BRIEF

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DR. MICHELLE Y. CHENG: “Our findings show that there are genes in the motor cortex that are both increased and decreased in animals that recovered spontaneously. Our hope is that we can target these genes in the cAMP signaling pathway and that it could one day be tested in patients to see if they could recover faster.”

RNA-sequencing data revealed a panel of genes associated with recovery in the motor cortex of mice who spontaneously recovered from stroke. The findings suggest therapeutic targets for stroke recovery in humans.

Some mice that have the same stroke injury recover better than others, a finding that led scientists at Stanford University School of Medicine to conduct RNA-sequencing analysis to see whether they could identify genes that play a role in recovery. They did.

In the study published in the September issue of Stroke, Michelle Y. Cheng, PhD, Gary K. Steinberg, MD, PhD, and their colleagues identified a panel of genes associated with recovery that are differentially expressed in the motor cortex. Ultimately, the finding could lead to new clues about how best to facilitate similar recovery in human stroke patients.

“To our knowledge, this is the first study to investigate the molecular signature in the motor cortex of stroke mice that naturally recovered, without external intervention,” said Dr. Cheng, a senior scientist in the department of neurosurgery at Stanford and co-senior author with Dr. Steinberg in the study.

Dr. Steinberg directs a stroke recovery lab, and several years ago Dr. Cheng and her colleagues began noticing that some of their animals had spontaneously recovered after stroke. “Understanding the genes regulated post-stroke could help us design novel ways to treat patients in the days and weeks after the initial event.”

STUDY METHODS, FINDINGS

For the study, seventy-five male mice between 11 and 13 days old underwent a transient middle cerebral artery occlusion. Thirty-three of the stroke animals and seven non-stroke sham animals were used in the analysis for the study.

The scientists mapped the stroke size and locations, and the animals were put through several behavioral tests over a two-week period. The scientists conducting the behavioral tests were blinded to whether the animals had a stroke or were in the sham surgery group. Before the stroke, animals were taught to balance on a horizontal rotating beam, and it took about 10 seconds to run the length of the slowly rotating beam. After the stroke injury, the animals usually cannot complete this task and fall off the beam. But two weeks into the behavioral tests, about 25 percent of them spontaneously recovered their ability to balance and walk on the beam without falling. The stroke size and location did not make a difference in those who recovered and those who did not.

On day 15 after the stroke, the animals were euthanized, and tissues were removed from the primary motor cortices on the side of the injury (ipsilesional) and the opposite hemisphere (contralesional) or non-stroke side. The researchers conducted RNA sequencing to see whether there were gene expression differences in those animals that spontaneously recovered and those that were not improving. (The stroke was not in the primary motor cortices, but the region plays a role in executing motor function.)

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DR. STEVEN C. CRAMER: “Gary Steinberg and his colleagues have asked some key questions and learned important answers that could have therapeutic implications. But this was in mice and mice are very different than humans. The chemistry of the brain changes day-to-day after stroke and this is a single snapshot. Now that we have a blueprint from mice, however, we can examine how these genes might inform approaches to enhance behavioral outcomes.”

They found major differences between the two groups. The gene expression analysis identified “distinct biological pathways in the spontaneously recovered stroke mice, in both ipsilesional and contralesional motor cortex,” said Dr. Cheng. They found 38 genes in the ipsilesional motor cortex that were significantly correlated with improved recovery, and 74 genes were correlated in the contralesional motor cortex. Many of these genes are involved with cAMP signaling in the contralesional motor cortex, with significant decreases in adenosine receptor A2A (Adora2a), dopamine receptor D2 (Drd2), and phosphodiesterase 10A (Pde10a) expression in recovered mice. The gene changes were correlated with the animal's behavior on the balancing beam.

“Our findings show that there are genes in the motor cortex that are both increased and decreased in animals that recovered spontaneously,” said Dr. Cheng. “Our hope is that we can target these genes in the cAMP signaling pathway and that it could one day be tested in patients to see if they could recover faster.

“The field has been focusing on what happens in the stroke and adjacent area. Now, scientists are focusing on changes in connected regions and the non-stroke contralesional side,” she added. “The genetic changes in the acute phase can be very different than the changes seen two weeks later. We are hoping to look at early and later time points to see if there is a transient or persistent change in gene expression.”

The Stanford group is now trying to understand the role of these genes on recovery. “We need to know the cell types regulating these genes, and then to see if we can manipulate them to promote recovery,” said Dr. Cheng.

Masaki Ito, MD, PhD, who along with Markus Aswendt, PhD, is co-first author on the study, had been working as a stroke neurosurgeon in Japan before joining the project for his post-doc. “There are still so many questions to answer,” he said. “What caused the difference between recovered and non-recovered animals? When and how long do we need to turn on the signaling to enhance recovery? How can we precisely and accurately modulate gene expression in this very small brain structure? These are next challenging steps.” Dr. Ito finished his post-doc and is back in Japan.

EXPERT COMMENTARY

“It's interesting that they can split the animals into two groups — recovered and non-recovered — and identify specific gene expression changes associated with the recovered group that are not seen in the non-recovered group,” said S. Thomas Carmichael, MD, PhD, the Frances Stark endowed chair and professor in the department of neurology at the David Geffen School of Medicine at UCLA and co-director of UCLA Broad Stem Cell Research Center. His group is also interested in the response of the brain that mediates recovery and the gene systems that led to recovery.

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TWO DISTINCT RECOVERY groups were identified using an analysis of the rotating beam behavior data. A novel lesion mapping analysis indicated that both the spontaneously recovered group and the non-recovered group exhibited a comparable lesion profile. RNA-sequencing transcriptome analysis of the ipsi- and contralesional primary motor cortices revealed a panel of recovery related genes in the spontaneously-recovered mice and highlighted the involvement of contralesional motor cortex in spontaneous recovery, particularly Adora2a, Drd2, and Pde10a-mediated cAMP signaling pathway.

One limitation, he said, is that the study captures only one point in time — two-weeks post-stroke — and “most rodents recover over six to eight weeks. This could have been a group of animals that just recovered faster or a true recovered/non-recovered response.”

Commenting on the study, Steven C. Cramer, MD, professor in the departments of neurology, anatomy & neurobiology, and physical medicine & rehabilitation at University of California, Irvine, said: “Gary Steinberg and his colleagues have asked some key questions and learned important answers that could have therapeutic implications. But this was in mice and mice are very different than humans. The chemistry of the brain changes day-to-day after stroke and this is a single snapshot. Now that we have a blueprint from mice, however, we can examine how these genes might inform approaches to enhance behavioral outcomes.”

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DR. S. THOMAS CARMICHAEL said its interesting that the researchers were able to “identify specific gene expression changes associated with the recovered group that are not seen in the non-recovered group.” But he said one limitation is that the study captures only one point in time — two-weeks post-stroke — and “most rodents recover over six to eight weeks. This could have been a group of animals that just recovered faster or a true recovered/non-recovered response.”

Craig E. Brown, PhD, MSFHR investigator and associate professor in the division of medical sciences at the University of Victoria, agreed. “This is a beautiful and simple idea that hasn't been studied before. They have identified a bunch of genes up- and down-regulated that are correlated with recovery. The next step is to manipulate gene expression in different cell types to see if they can enhance recovery. They have identified very interesting correlations which future studies can now use to provide more causal evidence.”

Rafael H. Llinas, MD, professor of neurology, director of neurology at Johns Hopkins Bayview, and a member of the Miller Coulson Academy of Clinical Excellence at Hopkins, added that “it would be difficult to manipulate the gene in such a way to boost recovery. But mechanistic studies like this teach us about the nature of recovery which is the first step to realizing treatments. It is likely that recovery studies in the past have failed due to our lack of a complete understanding of the underlying mechanisms of recovery.”

THE SCIENCE EXPLAINED: TRANSCRIPTOME ANALYSIS

WHAT IT IS: RNA sequencing transcriptome analysis is a high throughput gene expression technique that allows researchers to take a snapshot of actively expressed genes under specific conditions or in disease. Researchers can study the gene expression changes in a disease, profile thousands of genes at once, and identify the biological functions of these genes.

THE TECHNIQUE: RNA-sequencing allows researchers to measure gene expression. This high-throughput technique produces millions of sequences from complex RNA samples. The genes that are differentially expressed are analyzed with a software package that provides information on the significant canonical pathway, diseases, and functions. Quantitative real-time PCR is then used to validate the expression of the candidate genes between sham, non-recovered, and recovered stroke mice.

HOW IT IS USED: Transcriptome analysis enables the researchers to identify new genes or pathways that are involved in a disease, which leads to discovery of new genes that may be potential drug targets for treatment.

LINK UP FOR MORE INFORMATION:

•. Ito M, Aswendt M, Lee AG, et al. RNA-sequencing analysis revealed a distinct motor cortex transcriptome in spontaneously recovered mice after stroke https://www.ahajournals.org/doi/10.1161/STROKEAHA.118.021508. Stroke 2018; 49:2191–2199; Epub 2018 Aug 27.