KR102805604B1 - Method for suppressing sciatica by optogenetic stimulation of anteror cingulate cortex - Google Patents

Abstract

The present invention relates to a method for suppressing sciatica through optogenetic stimulation of neurons in the anterior cingulate cortex (ACC), and more specifically, to a method for suppressing sciatica through optogenetic stimulation, comprising the steps of: a 1st step of transforming neurons existing in the anterior cingulate cortex (ACC) of a mammal other than a human with a vector including a light-driven ion pump gene; a 2nd step of expressing the light-driven ion pump; and a 3rd step of irradiating the anterior cingulate cortex of the mammal with a light stimulus and inducing hyperpolarization of the neurons to suppress action potential generation, thereby inhibiting pain.
The method for suppressing sciatica of the present invention can reproduce the suppression of nerve cells in the anterior cingulate cortex (ACC) that appears in the pathological process of sciatica with a very high correlation with reality, and thus can be usefully utilized for the prevention or treatment of sciatica.

Description

{Method for suppressing sciatica by optogenetic stimulation of anteror cingulate cortex}

The present invention relates to a method for suppressing sciatica through optogenetic stimulation of neurons in the anterior cingulate cortex (ACC), and more specifically, to a method for suppressing sciatica through optogenetic stimulation, comprising the steps of: a 1st step of transforming neurons existing in the anterior cingulate cortex (ACC) of a mammal other than a human with a vector including a light-driven ion pump gene; a 2nd step of expressing the light-driven ion pump; and a 3rd step of irradiating the anterior cingulate cortex of the mammal with a light stimulus and inducing hyperpolarization of the neurons to suppress action potential generation, thereby inhibiting pain.

Pain is generally known to play an important role in protecting the body from danger and in the recovery of damaged tissue. However, pathological pain caused by infection with viruses or bacteria, severe inflammatory reactions around peripheral nerves, and direct damage to peripheral nerves is harmful to the human body.

In particular, neuropathic pain, which is known to be caused by damage to peripheral nerves, is accompanied by hyperalgesia (feeling more pain than it actually is) and allodynia (feeling even normal sensations as pain). Neuropathic pain can generally be caused by peripheral nerve damage (e.g., post-surgical pain or radiation), central nervous system damage (e.g., multiple sclerosis, spinal cord injury), viral infection (e.g., postherpetic neuralgia), tumors, etc. Neuropathic pain can be more severe than other types of chronic pain in terms of severity and duration, and 5% of patients complain of pain despite taking analgesics. Therefore, neuropathic pain is one of the most difficult clinical outcomes, and it cannot be controlled with standard pain medications and becomes resistant to numerous medications. The problem is that chronic neuropathic pain caused by peripheral nerve damage can be found to be refractory to current treatments.

Meanwhile, sciatica refers to pain along the areas (thighs, calves, feet, etc.) related to the sciatic nerve due to compression, damage, or inflammation of the sciatic nerve (sciatic nerve). Pain related to the sciatic nerve can be pain from the buttocks down to the thigh and leg, and may be accompanied by pain in the feet and toes.

Sciatica is reported to have a lifetime prevalence of 13-40%, and there is no difference in incidence between men and women. It is almost nonexistent in those under 20, but the incidence increases with age, peaking in those in their 40s, and decreasing in frequency after the 50s.

The sciatic nerve is formed by the 4th and 5th lumbar nerves and the 1st, 2nd, and 3rd sacral nerves. Herniated discs or spinal stenosis related to these lumbosacral nerves can cause symptoms. Excessive tension in the muscles around the sciatic nerve, as in piriformis syndrome, or myofascial pain syndrome can also cause symptoms.

There may be back pain, buttocks, back of thigh, calf or foot pain, and symptoms such as burning or tingling sensations, numbness, and weakness in the legs along with the pain. These symptoms may be caused by compression of the sciatic nerve by structures such as tumors or muscles through which the sciatic nerve passes, or by damage or inflammation of the sciatic nerve itself.

Although drug or surgical treatment is used to treat sciatica, there are still limitations to its use due to lack of satisfactory efficacy or side effects, and there is still a need for new surgical methods with improved efficacy and side effects.

Recently, as research on the brain has become more active, there have been reports that nerve cells in the anterior cingulate cortex (ACC) play an important role in the occurrence of neuropathic pain.

Accordingly, the inventors of the present invention have completed the present invention by providing a technology capable of suppressing pain by selectively controlling the activity of only the nerve cells in the prefrontal cortex, which are known to play a role in the generation of pain, by controlling the activity of the nerve cells through optical stimulation rather than electrical stimulation, without directly damaging the nerve cells.

Therefore, if the role of the neurons in the anterior cingulate cortex in the pathological process of pain can be more clearly identified and pain can be suppressed by artificially controlling them, it could be usefully utilized in research on the prevention or treatment of pain.

KR 10-1866252

Accordingly, the inventors of the present invention discovered through a series of experiments that the inhibition of neurons in the anterior cingulate cortex (ACC) is very important in the pathological process of sciatica, and based on this, made great efforts to develop an animal model capable of inhibiting pain response by artificially controlling the inhibition of neurons in the anterior cingulate cortex. As a result, they discovered that sciatica can be inhibited by transforming neurons in the anterior cingulate cortex of an animal with a light-driven ion pump gene and irradiating them with light stimulation, thereby completing the present invention.

Accordingly, the purpose of the present invention is to provide a method for suppressing sciatica through optogenetic stimulation, which comprises the following steps: 1) transforming neurons present in the anterior cingulate cortex (ACC) of a mammal other than a human with a vector containing a light-driven ion pump gene; 2) expressing the light-driven ion pump; and 3) irradiating the anterior cingulate cortex of the mammal with a light stimulus and suppressing the neurons to thereby inhibit pain.

To achieve the above purpose, the present invention provides a method for suppressing sciatica through optogenetic stimulation, including the following steps: 1) transforming neurons existing in the anterior cingulate cortex (ACC) of a mammal other than a human with a vector containing a light-driven ion pump gene; 2) expressing the light-driven ion pump; and 3) irradiating the anterior cingulate cortex of the mammal with a light stimulus to induce hyperpolarization of the neurons, thereby suppressing the generation of action potentials and inhibiting pain.

Optogenetics, used in the present invention, is a recently popular biotechnology field that manipulates genes to control something with light. Early research on optogenetics was applied to controlling the activity of nerve cells using light, which refers to a technology that activates nerve cells when they are exposed to light, and inhibits their activity when they are not exposed to light. Controlling the activity of nerve cells using light in this way allows for more precise control of nerve cell activity than controlling nerve cells using conventional electrical stimulation. This is because, while electrical stimulation stimulates an unspecified number of nerve cells, optogenetics technology can precisely stimulate specific target cells.

In the present invention, the light-driven ion pump is a membrane-permeable protein group that opens and closes pores by light stimulation. That is, when light is received, the channel of channelrhodopsin opens and something enters the cell, and when there is no light, the channel does not open. Functionally, halorhodopsin is a light-driven chloride pump that functions when exposed to yellow light with a wavelength of 580 nm to cause chloride ions to enter the cell, and through the above principle, when yellow light is shined on a nerve cell expressing halorhodopsin, hyperpolarization of the nerve cell is induced, thereby inhibiting the generation of action potentials.

In the present invention, the light-driven ion pump may be used without limitation as long as it is a protein that can open the ion pump of the cell membrane in response to exposure to a light stimulus, preferably halorhodopsin, and more preferably eNpHR3.0.

In the present invention, the method for transforming neurons into light-driven ion pumps can utilize any transformation method known in the art without limitation, and non-limiting examples thereof can be selected from the group consisting of transformation, transfection, electrophoresis, transduction, microinjection, and balliatic introduction, and more preferably, transfection, but is not limited thereto.

In one embodiment of the present invention, in order to specifically transfect neurons in the anterior cingulate cortex (ACC) with halorhodopsin (eNpHR3.0), an adenovirus vector expressing halorhodopsin (eNpHR3.0) was injected into the anterior cingulate cortex of a rat under the control of a neuron-specific hSyn promoter to express halorhodopsin (eNpHR3.0).

In the present invention, the vector may be selected from the group consisting of a linear DNA vector, a plasmid DNA vector, and a recombinant viral vector, but is not limited thereto, and all conventional vectors used for transformation in the art can be used in the method of the present invention.

The above three steps are the steps of irradiating the prefrontal cortex of mammals with light stimulation, inducing hyperpolarization of nerve cells, and inhibiting the generation of action potentials, thereby inhibiting pain.

In the present invention, it is preferable that the light stimulus is yellow light having a wavelength of 550 to 590 nm, and the time for irradiating the light stimulus is preferably 2 to 30 minutes. Meanwhile, the method for irradiating the light stimulus is not particularly limited, and the animal may be fixed and the light stimulus may be irradiated externally, or an optic fiber may be implanted into the prefrontal cortex to directly irradiate the light stimulus to the nerve cells in the prefrontal cortex. If the light stimulus irradiation time is less than 2 minutes, the problem of insufficiently inducing pain hypersensitivity in the animal may not occur, and if the light stimulus irradiation time exceeds 30 minutes, the pain hypersensitivity does not increase further, which is not preferable in terms of economy and time. The wavelength of the light may vary depending on the type of the light-driven ion pump to be transformed, and an ordinary technician can easily select the optimal light wavelength through repeated tests.

The method of suppressing sciatica through optogenetic stimulation of the present invention using an animal model of pain suppression by optogenetic stimulation of neurons in the prefrontal cortex can reproduce the suppression of neurons in the prefrontal cortex that appears in the pathological process of sciatica with a very high correlation with reality, and thus can be usefully utilized for the prevention or treatment of pain.

Figure 1 is a diagram showing the results of viral expression in the anterior cingulate cortex (ACC).
Figure 2 is a diagram showing the results of reducing mechanical and thermal sensitivity to neuropathic pain by light stimulation.
Figure 3 is a diagram showing the results of reducing mechanical and thermal sensitivity to neuropathic pain under various light stimulation conditions.

Hereinafter, the present invention will be described in detail with reference to the attached drawings as implementation examples of the present invention. However, the following implementation examples are presented as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description thereof may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express the preferred embodiment of the present invention, and this may vary depending on the intention of the user or operator, or the custom of the field to which the present invention belongs. Therefore, the definition of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a certain part is said to “include” a certain component, this does not mean that other components are excluded, but rather that other components can be included, unless specifically stated otherwise.

Although the present invention has been described in detail above only with respect to the described specific examples, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and it is natural that such modifications and variations fall within the scope of the appended claims.

Hereinafter, the present invention will be described in more detail through examples.

These examples are intended to explain the present invention more specifically, and the scope of the present invention is not limited to these examples.

-Experimental method-

Preparation Example 1. Establishment of an animal model of sciatica

1-1. Selection and design of experimental animals

All animal experiments were performed in accordance with approved national guidelines established by the Animal Experiment Ethics Review Committee of Chungbuk National University. Sterile animal surgical equipment, gloves, and sterile drapes and cotton swabs were used according to the guidelines of the International Association of Pain Studies. The skin was disinfected with 70% ethanol, and the fur was shaved from the surgical site, then covered with a sterile drape.

Sprague Dawley female rats (200-230 g, Coretech, Pyeongtaek, South Korea) were housed under standard housing conditions (12:12 h light/dark cycle) in accordance with the National Institutes of Health guidelines for animal welfare and had free access to food and water in the laboratory. Each rat was housed in a separate cage with flat paper bedding to prevent airway obstruction under random, double-blind, controlled guidelines.

1-2. Surgery for chronic constriction injury (CCI) of the sciatic nerve

In this study, CCI-treated and sham-treated (n = 10) mice were used as controls. All animals underwent surgery on the same day, and CCL-treated mice received peripheral nerve injury to the right hind limb under anesthesia to induce neuropathic pain. Cages were randomly selected from all pools and assigned to groups. Cages were temporarily numbered, and mice were numbered by the registry. All animals received the same anesthesia, preparation, sedation, and preoperative procedures, and animals were removed from their cages for the purpose of this study and permanently designated in the registry.

Preparation Example 2. Investigation of pain suppression through photo-stimulation in an animal model of sciatica

2-1. Virus injection for optogenetic techniques

To optogenetically stimulate neurons in the anterior cingulate cortex (ACC) and observe the resulting pain hypersensitivity, an adenoviral vector expressing eNpHR3.0 EYFP (halorhodopsin 3, yellow light-sensitive ion pump) under the control of the neuron-specific hSyn promoter was injected into the anterior cingulate cortex of mice (Ad-eNpHR3.0).

Simultaneously with CCI surgery, adenovirus vectors expressing halorhodopsin mutants or control adenoviruses expressing EYFP were injected into the anterior cingulate cortex (ACC) using a syringe pump (Legato® 130, KD Scientific, USA) at concentrations of 2.23 × 10^13 GC/ml and 1.9 × 10^13 GC/ml, respectively (human synapsin1(hSyn)-eNpHR3.0EYFP). Viral vectors were supplied by the Korea Institute of Science and Technology (Korea). Adenoviruses were injected at a rate of 0.4 μl/min.

The mice were divided into four groups according to the types of virus injection and surgery [i) group receiving optogenetic virus and CCI surgery: CCI/NpHR+, ii) group receiving NULL virus and CCI surgery: CCI/NpHR-, iii) group receiving optogenetic virus and SHAM: sham+NpHR+, iv) group receiving NULL virus and SHAM: sham+NpHR-).

The results of virus insertion were determined by observing the expression of c-fos immunoreactivity, a neural marker, to confirm the function of eNpHR. As shown in Fig. 1, in the CCL-treated group, the expression of c-fos in the prefrontal cortex was confirmed to increase in the group into which the NpHR virus was inserted compared to the group into which only the NULL virus was inserted.

2-2. Implantation of optical cannula in animal model injected with viral vector

Three weeks after the virus was injected according to the above method, a single-fiber optical cannula was implanted 2.5 mm below the skull for light transmission, and the optical fiber was inserted through the cannula so that it could reach the prefrontal cortex. Afterwards, laser-based light was irradiated to neurons expressing NpHR using the optical fiber.

Optical stimulation was performed using a DPSS 589 nm yellow laser (50 nm light irradiation and 20 Hz, 100 mW/mm2 ⁾ for 5 minutes via an optical fiber.

Preparation Example 3. Behavioral Experiment

3-1. Mechanical sensitivity

Mechanical hyperalgesia was measured with the dynamic plantar aesthesiometer (model 37450; Ugo Basile, Italy). Rats were placed in Plexiglas cages (20 × 20 × 14 cm) with a grid floor and allowed to rest for at least 30 min. Mechanical stimulation was applied by continuously increasing pressure (5 g/s) to the plantar aspect of the ipsilateral and contralateral CCI lesion, and changes in PWT (withdrawal threshold) and PWL (plantar hiding) were recorded.

3-2. Thermal sensitivity

Thermal pain sensitivity was measured using a Peltier plate (hot/cold plate, Ugo Basile, Italy). For the thermal sensitivity test, rats were placed on a temperature-controlled plate set at 50 °C, and nociceptive behaviors such as paw flicking or licking were recorded as responses.

-Experimental Results-

Experimental Example 1. Changes in pain behavior due to sciatica

To evaluate the sensitivity to neuropathic pain in CCI-induced and control groups (SHAM), behavioral analysis was performed to find PWT and PWL. Physical and thermal sensitivity were increased, and PWT, PWL, and thermal latency were compared among the groups.

For the CCI group, the PWT ranged from 35.20±2.53 to 34.75±7.27 g, while in the control group (SHAM), the ranged from 36.075±5.09 to 12.98±3.22 g, showing a significant difference (Fig. 2a).

In addition, for the CCL group, it was confirmed that the PWL had a range of values from 20.39±3.22 to 6.92±2.69 s, while for the control group (SHAM) it had a range of values from 19.45±2.36 to 17.78±2.50 s (Fig. 2b). Finally, for the CCL group, it was confirmed that the thermal latency had a range of values from 20.99±5.72 to 9.08±2.73 s, while for the control group (SHAM) it had a range of values from 20.73±5.50 to 20.46±2.94 s (Fig. 2c).

animal group basic Day1 Day7 Day14 Day21 Paw withdrawal threshold (in grams) CCI ipsilateral 36.07 ± 5.09 12.03±2.62* 14.95±4.03* 13.98±4.56* 12.98±3.22* CCI contralateral 36.50 ± 5.19 30.33 ± 6.99 35.48 ± 4.52 35.43 ± 4.18 35.25 ± 4.98 Sham ipsilateral 35.20 ± 2.53 32.10 ± 3.92 33.49 ± 4.20 33.65 ± 5.33 34.75 ± 7.27 Sham contralateral 36.34 ± 7.27 34.12 ± 4.67 34.02 ± 7.46 35.21 ± 2.93 35.25 ± 4.44 Paw withdrawal latency (in seconds) CCI ipsilateral 20.39 ± 3.22 5.90 ± 2.00* 6.93 ± 1.30* 7.63 ± 4.26* 6.92 ± 2.69* CCI contralateral 20.85 ± 4.48 16.76 ± 2.75 20.49 ± 4.46 18.27 ± 2.46 20.04 ± 3.32 Sham ipsilateral 19.46 ± 2.36 17.09 ± 2.44 17.55 ± 3.91 17.35 ± 2.97 17.78 ± 2.50 Sham contralateral 19.14 ± 3.61 18.86 ± 3.27 18.66 ± 4.00 18.03 ± 3.80 18.66 ± 2.77 Thermal latency (in seconds) CCI 20.99 ± 5.72 10.69±2.18* 10.32±2.51* 9.17 ± 2.63* 9.08 ± 2.73* Sham 20.73 ± 5.50 19.77 ± 3.17 20.82 ± 3.64 20.37 ± 2.25 20.46 ± 2.94

Experimental example 2. Pain relief by optogenetic stimulation of neurons in the anterior cingulate cortex (ACC)

In the group that received optogenetic virus and CCI surgery (CCI/NpHR+), PWT [before treatment: 7.05±2.87 (mean±standard deviation), upon yellow light irradiation: 29.7±3.42 (mean±standard deviation), after treatment: 12.62±4.73 (mean±standard deviation)] significantly increased when stimulated with yellow light (589 nm), and PWL [before treatment: 5.2±2.67 (mean±standard deviation), upon yellow light irradiation: 16.42±4.20 (mean±standard deviation), after treatment: 5.52±1.74 (mean±standard deviation)] also showed a significant improvement effect when stimulated with yellow light (see Fig. 3).

That is, in the case of yellow light irradiation, the CCL-treated group that received NpHR showed antinociceptive behavior compared to the other groups. These results confirmed that cells expressing NpHR are triggered by yellow light of a specific wavelength, which induces hyperpolarization of neurons, contributing to inhibition and analgesia of neurons.

animal group Pre Stim Post Paw withdrawal threshold (in grams) CCI/NpHR+ ipsi 7.05 ± 2.87 29.7 ± 3.42* 12.62 ± 4.73 Contra 32.7 ± 5.58 35.75 ± 4.78 36.85 ± 4.78 CCI/NpHR- ipsi 12.95 ± 2.32 11.52 ± 4.58 12.47 ± 4.16 Contra 37.07 ± 5.01 37.45 ± 5.01 34.6 ± 5.38 Sham NpHR+ ipsi 38.85 ± 6.95 35.25 ± 2.75 32.7 ± 0.2 Contra 37.4 ± 1.7 35.75 ± 0.55 37.2 ± 2 Sham NpHR- ipsi 33.05 ± 4.85 36 ± 3.5 40.8 ± 0.6 Contra 37.4 ± 0.8 36 ± 0.8 37.8 ± 4.3 Paw withdrawal latency (in seconds) CCI/NpHR+ ipsi 5.2 ± 2.67 16.42 ± 4.20* 5.52 ± 1.74 Contra 16.75 ± 3.18 17.85 ± 2.44 18.02 ± 2.82 CCI/NpHR- ipsi 6.15 ± 1.90 5.92 ± 1.84 7.72 ± 1.00 Contra 17.57 ± 3.36 18.77 ± 2.51 17.35 ± 2.34 Sham NpHR+ ipsi 20.9 ± 2 22.45 ± 3.75 19.9 ± 3.7 Contra 18.55 ± 1.25 17.35 ± 0.75 17.95 ± 1.85 Sham NpHR- ipsi 18.05 ± 1.65 20.8 ± 2.1 23.45 ± 2.75 Contra 17.3 ± 0.9 18.05 ± 0.85 19.45 ± 1.95 Thermal latency (in seconds) CCI/NpHR+ 7.52 ± 1.46 15.95 ± 2.08* 8.17 ± 1.29 CCI/NpHR- 8.32 ± 1.14 7.9 ± 0.86 7.05 ± 1.20 Sham NpHR+ 21 ± 2.1 20.7 ± 2 18.7 ± 1.9 Sham NpHR- 22.35 ± 2.55 19.65 ± 1.15 21.65 ± 0.95

Although the present invention has been described in detail above only with respect to the described specific examples, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and it is natural that such modifications and variations fall within the scope of the appended claims.

Claims (5)

Step 1: Transforming neurons in the anterior cingulate cortex (ACC) of mammals other than humans with a vector containing a light-driven ion pump gene;
Step 2 of expressing the above light-driven ion pump; and
A method for suppressing sciatica through optogenetic stimulation, comprising three steps of irradiating the prefrontal cortex of the mammal with light stimulation, inducing hyperpolarization of neurons, and suppressing action potential generation to suppress pain.
The above light-driven ion pump is eNpHR3.0.
An inhibition method, characterized in that the above light stimulation is irradiated with light having a wavelength of 550 to 590 nm for 2 to 8 minutes. delete delete A method for suppressing sciatica through optogenetic stimulation, characterized in that in claim 1, the vector is selected from the group consisting of a linear DNA vector, a plasmid DNA vector, and a recombinant viral vector. delete