Surgery Godfather

Chapter 2188: Chapter 1434: Crossing the River by Feeling the Stones (Part 2)

"But if we don’t do it," Yang Ping looked at him, "M7 may suffer from chronic pain while regaining motor functions. Would you want to see it trembling in pain while walking? Before having an effective solution at the molecular level, this is the best approach."

Tang Shun opened his mouth but said nothing.

Weber broke the silence, "Professor Yang, theoretically, your plan is valid. But the application of optogenetics in primate spinal cords has no precedent. We need to conduct a lot of pre-experiments to verify the targeting of the virus vector, the depth of light stimulation, and long-term safety."

"I know," Yang Ping said, "so we proceed in two steps. The first step is to verify in vitro, testing the transfection efficiency of the virus vector and the effect of light stimulation on spinal cord tissue sections of M7. The second step, if successful in vitro, then conduct in vivo experiments on M7."

"What about the timeline?" Eva asked, "According to your schedule, we have at most a two to three-week window."

"The in vitro verification will take a week," Yang Ping calculated, "if successful, the in vivo experiments require two to three days of preparation, the implantation can be completed within a week. Just in time."

"What if it fails?" Manstein asked.

Yang Ping was silent for a few seconds, then said, "Then we use the alternative, intrathecal injection of Baclofen, a GABA_B receptor agonist. The effect is not as precise as optogenetics but can enhance inhibitory regulation systemically. The side effect is reduced muscle tone, which may affect motor recovery."

"So optogenetics is Plan A, and Baclofen is Plan B," Weber summarized, "I agree with this plan. But with one condition, the in vitro verification must be done by me personally, optogenetics is my specialty. I conducted similar experiments in Heidelberg."

"Alright," Yang Ping nodded, "Professor Weber will handle the in vitro verification, Eva will upgrade the closed-loop sensor, Lina will handle the data analysis model, Hans will prepare the virus vector and optical fibers. Tang Shun, you coordinate all resources to ensure results within a week."

"Understood!"

The in vitro verification begins.

Weber personally performed the surgery, extracting a small tissue piece, about two cubic millimeters, from M7’s spinal injury area, placed it in icy artificial cerebrospinal fluid, and sliced it into thin sections 300 micrometers thick. These slices were transferred to a culture dish, maintained in a viable state, capable of recording electrophysiological activities for four to six hours continuously.

The virus vector is AAV9-hSyn-NpHR-EYFP, a modified adeno-associated virus. AAV9 has a high affinity for neurons, hSyn promoter ensures NpHR is expressed only in neurons, and EYFP serves as a fluorescent marker, facilitating the observation of transfection efficiency under the microscope.

Weber dripped the virus solution onto the spinal cord sections, incubating for two hours. Then he washed them three times with PBS before observing under a confocal microscope.

"Transfection efficiency," he adjusted the focus, "approximately 35%."

"35%," Manstein recorded beside him, "is it enough?"

"Theoretically, the inhibitory interneurons in the spinal cord dorsal horn account for 20 to 30% of the total neuron count. A 35% transfection efficiency means most inhibitory neurons can be targeted, it’s sufficient."

The next step is electrophysiological verification, Weber placed a transfected spinal cord section in the electrophysiological recording slot, using glass microelectrodes to record the spontaneous firing of dorsal horn neurons. Simultaneously, he placed an optical fiber with a diameter of 200 micrometers above the section, connected to a yellow laser.

"Baseline firing frequency," he looked at the oscilloscope, "five hertz per second, within the normal range."

Then he turned on the laser. A faint yellow light passed through the optical fiber, illuminating the surface of the spinal cord section.

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

The waveform on the oscilloscope changed, spontaneous firing frequency dropped from five hertz per second to one hertz per second, a decrease of 80%.

"Inhibitory effect confirmed," Weber’s voice was calm, but Manstein noticed his hand trembled slightly, "After NpHR activation, chloride ion influx, neurons hyperpolarize, firing reduced."

"Is it reversible?" Yang Ping asked.

Weber turned off the laser, ten seconds later, the firing frequency returned to 4.8 hertz per second.

"Completely reversible."

A restrained cheer erupted in the meeting room. Tang Shun and Hans clapped hands, Lina rapidly typed on her laptop, Eva rarely showed a trace of a smile.

But Weber did not smile, he stared at the spinal cord section for a long time, then said, "This is just in vitro, the in vivo environment is far more complex, inflammation, immune responses, glial scarring, blood flow changes. In vitro success does not mean in vivo will succeed."

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

"Crossing a river by feeling the stones?"

Weber pondered this statement, he praised:

"That’s a philosophical saying."

Immediately, the in vivo experiment also commenced.

This time, the scale of the surgery was larger than the previous two. Yang Ping was the chief surgeon, Manstein assisted, Eva was responsible for electrophysiological monitoring, Hans handled virus injection and optical fiber implantation, Weber observed from below the stage.

The first step of the surgery was virus injection. Hans used a glass microinjection needle to slowly inject five microliters of AAV9-hSyn-NpHR-EYFP solution into M7’s spinal cord dorsal horn, bilaterally, one microliter each injection site, totaling ten injection sites. The injection speed was extremely slow, 0.1 microliters per minute, to prevent pressure damage.

"Virus injection completed," Hans reported, "wait fifteen minutes for the virus to spread."

Fifteen minutes later, the second step, optical fiber implantation. Eva designed a set of miniature optical fiber arrays, four optical fibers, each with a diameter of 200 micrometers, arranged in a diamond shape, covering the main area of the spinal cord dorsal horn. The ends of the optical fibers connected to a subcutaneously implanted LED module, powered by wireless charging, adjustable light intensity and frequency via an external controller.

"Optical fiber implantation completed," Yang Ping said, "close the incision."

The surgery lasted over an hour. When M7 woke from anesthesia, its hind limb muscle strength hadn’t changed, the virus and optical fibers themselves would not affect motor functions. But everyone knew that the real test had not yet begun.

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

During the waiting days, the institute’s atmosphere was tense like a taut string. Every morning, Eva would perform electrophysiological monitoring on M7, recording spontaneous firing frequencies in the spinal cord dorsal horn. For the first three days, there were no significant changes in the data, NpHR had not expressed sufficiently yet.

The fourth day, changes appeared.

M7’s dorsal horn spontaneous firing frequency suddenly rose from the normal three to five hertz per second to twelve hertz per second. This is a dangerous signal, abnormal high-frequency firing indicates central sensitization forming.

"Initiate light stimulation," Weber said.

Eva pressed the controller’s button, yellow light passed through the optical fibers, shining into M7’s spinal cord dorsal horn.

Ten seconds later, firing frequency fell to six hertz per second. Twenty seconds later, it dropped to four hertz per second. A minute later, stabilized at 3.5 hertz per second, lower than baseline, indicating good inhibitory effect.

"Effective," Weber’s voice conveyed a slight tremor, "Optogenetic closed-loop regulation, effective."

Yang Ping exhaled deeply, looking at the small yellow indicator light on the controller, thinking it was the most beautiful light he had ever seen.

Indeed, scientific research is like this, crossing a river by feeling the stones, no one knows what the outcome will be, but adjustments are made along the way, slowly a path is cleared. Without a bold start, there would never be a correct path.

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