godfather of surgery

Chapter 1434 Crossing the River by Feeling the Stones

Chapter 1434 Crossing the River by Feeling the Stones

The results of the cotton test were unexpected by everyone.

Fritz started with a cotton ball on the back of M7's foot and moved it upwards little by little. The high-speed camera recorded at a speed of one thousand frames per second, capturing even the slightest muscle tremor that was invisible to the naked eye in slow motion.

“Instep, no response,” Eva stared at the live replay screen. “Ankle, no response. Lower leg… wait, stop.”

She pressed the pause button, rewound the video by three frames, and then zoomed in to the pixel level.

On the skin of M7's mid-calf, an almost invisible ripple appeared. It wasn't a muscle contraction, but a tremor of the skin itself, like the slightest ripple when the wind blows across a lake. Fritz's cotton ball had just brushed past there, the force so light it was almost imperceptible.

“This is a tactile response,” Eva’s voice was slightly strained, “Aβ fiber-mediated light touch, with an extremely low threshold. The light touch threshold in normal primates is between 0.5 and 1 gram; this stimulus was probably less than 0.2 grams.”

"What does that mean?" Yang Ping asked.

“This illustrates two points,” Eva said, pulling up previous pain threshold data. “First, the sensory pathways in M7 are indeed recovering, and the order of recovery matches theoretical predictions—pain recovers first, then light touch, and finally proprioception. Second, its tactile threshold is lower than normal, meaning there may be tactile hypersensitivity, which shares the same mechanism as the previous pain hypersensitivity—central sensitization.”

Weber walked over and looked at the almost invisible ripple on the screen.

“Central sensitization,” he repeated, “is one of the most common complications after spinal cord injury. It’s not that patients can’t feel anything, it’s that they feel too much. Normal touch becomes painful, normal temperature becomes burning.”

“Yes,” Yang Ping nodded, “and once central sensitization occurs, it’s very difficult to reverse. It involves changes in synaptic plasticity of neurons in the dorsal horn of the spinal cord, NMDA receptor activation, calcium ion influx, and then a series of cascade reactions: the MAPK pathway, CREB phosphorylation, and altered gene expression. It’s a positive feedback loop: the more sensitized, the more excited; the more excited, the more sensitized.”

“So we face a paradox,” Mainstein frowned, “the protocellular activation strategy promotes neurogenesis, but the regenerated nerves may carry ‘wrong signals,’ leading to central sensitization. We cure paralysis, but we may create a patient in pain.”

Silence fell over the meeting room once again.

“No,” Yang Ping suddenly said, “it’s not a paradox, it’s a matter of time windows.”

He walked to the whiteboard and drew a timeline.

"The formation of central sensitization requires two conditions: first, abnormal neural input, where residual or regenerated nerve fibers after injury fire abnormal impulses; second, changes in the plasticity of neurons in the dorsal horn of the spinal cord, with increased synaptic strength and decreased function of inhibitory interneurons. Both of these conditions require time."

He marked two points on the timeline: "In the third week after surgery, pain sensation returns, which is the first stage of abnormal afferent input; in the fourth week after surgery, tactile sensation returns. If left untreated, central sensitization will solidify in another two to three weeks."

“But now we know,” he said, pointing to the fourth week, “that we can intervene within this time window to prevent the sensitization from solidifying.”

"How should we intervene?" Tang Shun asked.

“Two approaches,” Yang Ping put down his pen. “The first is to reduce abnormal input. For example, using sodium channel blockers to inhibit ectopic discharges in damaged nerve fibers. However, sodium channel blockers can affect normal nerve conduction, potentially negating our recovery efforts.”

"What about the second one?"

“Enhancing inhibitory regulation,” Yang Ping continued, “is crucial. The dorsal horn of the spinal cord contains a large number of inhibitory interneurons, primarily GABAergic and glycineergic. A key mechanism of central sensitization is the decline in the function of these inhibitory neurons. If we can enhance their activity, we can block the positive feedback loop of sensitization without affecting normal afferent input.”

“GABA receptor agonists?” Weber asked.

"Yes, but not just simple agonists. GABA_A receptors are chloride ion channels, and continuous activation leads to receptor desensitization, resulting in progressively weaker effects. What we need is a 'smart' regulatory strategy that enhances inhibition only during periods of abnormal excitation, while remaining unaffected during normal activity."

“This sounds like…” Lina pushed up her glasses.

“Closed-loop neural regulation,” Eva continued, “is based on the same idea as my biosensor. It monitors the electrical activity of the dorsal horn of the spinal cord in real time, and automatically releases GABAergic agonists when abnormally high-frequency discharges are detected; it does not release them during normal discharges.”

“But how can GABAergic agonists be released locally?” Hans frowned. “Systemic administration can cross the blood-brain barrier, leading to sedation and cognitive impairment. Local administration requires a pump, which carries a high risk of infection.”

“We don’t need exogenous drugs,” Yang Ping shook his head. “We can use optogenetics.”

“Optogenetics?” Mainstein’s eyes widened. “In primates?”

“Yes,” Yang Ping nodded. “In 2016, a team conducted optogenetic experiments on the visual cortex of rhesus monkeys, proving its safety and feasibility. Our approach is to use a viral vector to introduce light-sensitive ion channels, such as NpHR, a chloride ion pump, into inhibitory interneurons in the dorsal horn of the M7 spinal cord. Then, using an implanted optical fiber, when abnormal electrical activity is detected, yellow light is emitted to activate the NpHR, enhancing local inhibition.”

"This is too advanced," Tang Shun gasped. "Viral vectors, optogenetics, closed-loop regulation—these three technologies combined are catastrophic if any one of them goes wrong."

“But if we don’t do it,” Yang Ping looked at him, “M7 might suffer from chronic pain while regaining motor function. Would you want to see it walking while trembling in pain? Until an effective solution is found at the molecular level, this is the best approach.”

Tang Shun opened his mouth, but didn't say anything.

Weber broke the silence: "Professor Yang, your plan is theoretically sound. However, there's no precedent for applying optogenetics in the primate spinal cord. We need to conduct extensive preliminary experiments to verify the targeting of the viral vector, the depth of light stimulation, and its long-term safety."

“I know,” Yang Ping said, “so we’ll proceed in two steps. First, we’ll conduct in vitro validation, testing the transfection efficiency of the viral vector and the effect of light stimulation on spinal cord slices from M7. Second, if successful in vitro, we’ll perform in vivo experiments on M7.”

“What about the timeline?” Eva asked. “According to your timeline, we have at most two to three weeks left.”

“In vitro validation will take a week,” Yang Ping calculated. “If successful, in vivo testing will require two to three days of preparation, and implantation can be completed within a week. Just in time.”

“What if we fail?” Mainstein asked.

Yang Ping paused for a few seconds, then said, "Then let's use the alternative: intrathecal injection of baclofen, a GABA_B receptor agonist. The effect isn't as precise as optogenetics, but it can systemically enhance inhibitory regulation. The side effect is decreased muscle tone, which may affect motor recovery."

“So optogenetics is Plan A, and baclofen is Plan B,” Weber concluded. “I agree to this plan. But there’s one condition: the in vitro validation must be done by me personally. Optogenetics is my forte; I did similar experiments back in Heidelberg.”

“Okay,” Yang Ping nodded. “Professor Weber will be in charge of in vitro validation, Eva will be in charge of the closed-loop sensor upgrade, Lina will be in charge of the data analysis model, and Hans will be in charge of the viral vector and fiber optic preparation. Tang Shun, you coordinate all resources to ensure results are available within a week.” “Understood!”

In vitro validation begins.

Weber personally performed the surgery, taking a small tissue sample, approximately two cubic millimeters in size, from the spinal cord injury area of ​​M7. This sample was placed in cold, artificial cerebrospinal fluid and sliced ​​into thin sections three hundred micrometers thick. These sections were then transferred to petri dishes to maintain their viability, allowing for continuous recording of electrophysiological activity for four to six hours.

The viral vector was AAV9-hSyn-NpHR-EYFP, a modified adeno-associated virus. AAV9 exhibits high tropism for neurons, the hSyn promoter ensures that NpHR is expressed only in neurons, and EYFP serves as a fluorescent label, facilitating microscopic observation of transfection efficiency.

Weber placed the virus solution onto a spinal cord slice and incubated it for two hours. He then rinsed the slice three times with PBS and observed it under a confocal microscope.

“Transfection efficiency,” he adjusted the focus, “approximately thirty-five percent.”

“Thirty-five percent,” Mainstein noted down beside him, “Is that enough?”

"Theoretically, inhibitory interneurons in the dorsal horn of the spinal cord account for 20% to 30% of the total number of neurons. A 35% transfection efficiency means that most inhibitory neurons can be targeted, which is sufficient."

The next step was electrophysiological verification. Weber placed a transfected spinal cord slice in an electrophysiological recording chamber and used glass microelectrodes to record the spontaneous firing of dorsal horn neurons. At the same time, he placed a 200-micrometer-diameter optical fiber above the slice and connected it to a yellow laser.

“Baseline discharge frequency,” he said, looking at the oscilloscope, “five hertz per second, within the normal range.”

Then he turned on the laser. A faint beam of yellow light shone through the optical fiber onto the surface of the spinal cord slice.

"Light intensity, one milliwatt per square millimeter; wavelength, 590 nanometers."

The waveform on the oscilloscope changed; the spontaneous discharge frequency dropped from five hertz per second to one hertz per second, a decrease of eighty percent.

“The inhibitory effect is confirmed,” Weber’s voice was calm, but Mainstein noticed his hands trembling slightly. “After NpHR is activated, chloride ions flow inward, neurons hyperpolarize, and firing decreases.”

"Is it reversible?" Yang Ping asked.

Weber turned off the laser, and ten seconds later, the discharge frequency returned to 4.8 Hz per second.

"Completely reversible."

A suppressed cheer erupted in the conference room. Tang Shun and Hans high-fived, Lina typed rapidly on her notebook, and Eva gave a rare, slight smile.

But Weber didn't laugh. He stared at the spinal cord slice for a long time, then said, "This is only in vitro. The internal environment is much more complex: inflammation, immune responses, glial scarring, changes in blood flow. Success in vitro doesn't guarantee success in vivo."

“I know,” Yang Ping said, “but at least we’ve proven that this direction is correct; scientific research is like crossing a river by feeling for stones.”

"Feeling one's way across the river by touching the stones?"

Weber pondered this statement, and he exclaimed in admiration:

"This is a very philosophical statement."

Immediately afterwards, in vivo experiments also began.

This time, the surgery was larger in scale than the previous two. Yang Ping was the lead surgeon, with Mainstein assisting, Eva in charge of electrophysiological monitoring, Hans in charge of virus injection and fiber optic implantation, and Weber observing from the sidelines.

The first step of the surgery was viral injection. Using a glass microneedle, Hans slowly injected five microliters of AAV9-hSyn-NpHR-EYFP solution into the dorsal horn of the M7 spinal cord, bilaterally, one microliter at each injection point, for a total of ten injection points. The injection rate was extremely slow, at 0.1 microliters per minute, to avoid pressure damage.

"Virus injection complete," Hans reported. "Wait fifteen minutes for the virus to spread."

Fifteen minutes later, the second step was fiber optic implantation. Eva designed a miniature fiber optic array consisting of four fibers, each 200 micrometers in diameter, arranged in a rhombus to cover the main area of ​​the dorsal horn of the spinal cord. The ends of the fibers were connected to a subcutaneously implanted LED module, which was wirelessly charged and whose light intensity and frequency could be adjusted via an external controller.

"Fiber optic implantation complete," Yang Ping said. "The incision closed."

The surgery lasted for over an hour. When M7 awoke from anesthesia, its hind limb muscle strength remained unchanged; the virus and fiber optic cable themselves did not affect its motor function. But everyone knew the real test was yet to come.

NpHR expression takes time. After the virus enters neurons, it takes three to five days to express enough protein. Therefore, the first half of the fifth week after surgery is a waiting period.

During the waiting period, the atmosphere at the research institute was tense, like a taut string. Every morning, Eva would perform electrophysiological monitoring on M7, recording the spontaneous discharge frequency of the spinal cord dorsal horn. For the first three days, the data showed no significant changes; NpHR was not yet sufficiently expressed.

On the fourth day, a change occurred.

The spontaneous discharge frequency of M7 at the back angle suddenly increased from the normal three to five hertz per second to twelve hertz per second. This is a dangerous signal; the abnormally high frequency discharge means that central sensitization is forming.

“Activate light stimulation,” Weber said.

Eva pressed the button on the controller, and yellow light shone through the optical fiber into the dorsal horn of M7's spinal cord.

Ten seconds later, the discharge frequency dropped to six hertz per second. Twenty seconds later, it dropped to four hertz per second. One minute later, it stabilized at three and a half hertz per second, lower than the baseline, indicating good suppression.

“Effective,” Weber’s voice came through, trembling slightly. “Closed-loop regulation of optogenetics, effective.”

Yang Ping let out a long sigh of relief. He looked at the small yellow indicator light on the controller and thought it was the most beautiful light he had ever seen.

That's how scientific research works; it's like crossing a river by feeling for stones. No one knows what the outcome will be; you can only adjust as you go, and slowly you'll find a path. Without a bold start, you'll never find the right way.