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Mar 2021
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Cortical Laminar Recording of Multi-unit Response to Distal Forelimb Electrical Stimulation in Rats
大鼠前肢远端电刺激的多单位反应的皮层层析记录   

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Abstract

Severe traumatic brain injury (sTBI) survivors experience permanent functional disabilities due to significant volume loss and the brain’s poor capacity to regenerate. Chondroitin sulfate glycosaminoglycans (CS-GAGs) are key regulators of growth factor signaling and neural stem cell homeostasis in the brain. In this protocol, we describe how to perform recordings to quantify the neuroprotective and regenerative effect of implanted engineered CS-GAG hydrogel (eCS) on brain tissue. This experiment was performed in rats under three conditions: healthy without injury (Sham), controlled cortical impact (CCI) injury on the rostral forelimb area (RFA), and CCI-RFA with eCS implants. This protocol describes the procedure used to perform the craniotomy, the positioning of the cortical recording electrode, the positioning of the stimulation electrode (contralateral paw), and the recording procedure. In addition, a description of the exact electrical setup is provided. This protocol details the recordings in the brain of injured animals while preserving most of the uninjured tissue intact, with additional considerations for intralesional and laminar recordings of multi-unit response.


Graphic abstract:


Sensorimotor response to paw stimulation using cortical laminar recordings.


Keywords: Linear silicone probe (线性硅胶探头), Caudal forelimb area (尾前肢区), Paw stimulation (爪子刺激), Laminar cortical recording (层状皮质记录), Multi-unit sensory response (多单元感官反应)

Background

Traumatic brain injury (TBI) is a common and increasingly prevalent problem that affects approximately 69 million people globally, without an effective treatment to date (Dewan et al., 2018). Given the failure of secondary neuroprotective strategies, such as decompressive craniotomy, or tight blood pressure regulation, in ameliorating poor functional outcomes, increased attention has turned to re-establishing damaged neuronal circuitry in the brain through biomaterial implants, with or without concurrent cell transplantation (Tan et al., 2020). Apart from providing the ability to inject any one of several natural or synthetic biomaterials into an injury lesion, it is unclear what functional effect these implants have on restoring native neuronal circuits and on higher-order cognitive and motor outcomes. In our manuscript (Latchoumane et al., 2021, DOI: 10.1126/sciadv.abe0207), we implanted rats with engineered chondroitin-sulfate glycosaminoglycans (eCS) as a potential treatment for the loss of tissue and consequent loss of motor function following TBI. To assess the physiological recovery promoted by eCS implants, we recorded the laminar cortical activity in response to electrical stimulation of the paw in anesthetized rats. Previous works in the field have used ex vivo planar multielectrode arrays on 300 µm brain slices to evaluate field excitatory postsynaptic potentials (fEPSPs) post-biomaterial implant (Yang et al., 2015; Hao et al., 2017). Other labs have performed steady-state evoked potentials (SSEP) recordings with chronically implanted electrodes in the mouse brain to measure brain responses post-implant (Fernández-García et al., 2016). We present a simple protocol that can be carried out in a single procedure, using a multichannel system recording and stimulation setup for the evaluation of the sensorimotor integration in animals implanted with eCS following TBI. Our protocol demonstrates the feasibility and reproducibility of recording perilesionnally to assess biomaterial integration, the impact of eCS on surrounding tissue, and the extent of live neuronal proliferation within and around the implant.

Materials and Reagents

  1. Suture 4-0 Ethicon Absorbable plus antibacterial (Vicryl, catalog number: 109162)

  2. Self tapping screws (18-8 Stainless Steel Slotted Flat Head Screws, M0.8 × 0.2 mm Thread, 2 mm Long; McMastercarr, catalog number: 91430A143)

  3. Sterile cotton swabs, Cotton Tipped Applicators 6"/Sterile 100/box (Dynarex)

  4. Stimulation needles, sterile stainless steel needle 24 gauge (BD, Microlance, catalog number: 1730738)

  5. Animals: Sprague-Dawley Rats, male, age (7-10 weeks) (Charles River, catalog number: 400)

  6. 32 channel linear probe (Neuronexus, A1x32-6mm-50-177-CM32, 15 µm thickness, Gen4, lot# P994)

  7. Ketamine 100 mg/ml (Coventrus, catalog number: 056347361-4)

  8. Xylazine cocktail, 100mg/ml (Sigma-Aldrich, catalog number: 7361-61-7)

  9. Isofluorane (Coventrus, catalog number: 029405 )

  10. Buprenorphine, 0.03 mg/ml (Coventrus, catalog number: 059122)

  11. Marcaine, 0.5% (Coventrus, catalog number: 054893)

  12. Povidone-Iodine, 10% topical solution (CVS, catalog number: 59779-085)

  13. Etch-Gel, phosphoric acid 40% (DMG, catalog number: 61901)

  14. Gel Foam® (Pfizer, catalog number: 115631)

  15. SeaKem® agarose (Lonza, catalog number: 50004)

  16. Triple antibiotics, 0.9 g Pouch (25 ct. Box) (Safetec, catalog number: 53205)

  17. Ketamine/Xylazine Cocktail (see Recipes)


Ketamine/Xylazine Cocktail:

  1. For 1 ml solution: 0.9 ml of Ketamine (100 mg/ml) + 0.1 ml of Xylazine (100 mg/ml)

  2. KX rat cocktail 0.1 ml/100g rat wt. IP (Ketamine: 90 mg/kg, Xylazine: 9.0 mg/kg)

Equipment

  1. Multichannel acquisition system (MCS, Wireless Recording, model: W2100)

  2. Multichannel recording and stimulation headstage (MCS, headstage, model: HS32-EXT0.5mA)

  3. Dell PC i7, RAM:8Go, SSD: 500 GoPremium Silicone

  4. Kopf Stereotaxic frame for rodent with manipulating arm

  5. Electronic breadboard (Half-size breadboard; 63; adafruit.com)

  6. Electronic cables (Covered Male-Male Jumper Wires, 200 mm × 40; Adafruit.com, catalog number: 4482)

  7. MCS coaxial TTL cable (C-BNC-Lemo1m; Multichannel system accessories)

  8. Electric Shaver (Philips, Norelco oneblade QP2520/90)

  9. Electric Drill and trephine bur (Micromotor shiyang, H102S)

  10. CCI tip, 3 mm diameter (Custom made)

  11. CCI impactor machine (UGA workshop custom made)

Software

  1. Acquisition Software: MCS experimenter (Multichannel Systems, https://www.multichannelsystems.com/)

  2. System configuration: MCS IPconfig (Multichannel Systems, https://www.multichannelsystems.com/)

  3. Data analysis: Matlab + MCS toolbox (Multichannel Systems, https://www.multichannelsystems.com/)

  4. Matlab R2019b (Mathworks Inc., mathworks.com)

Procedure

  1. System setup

    The system setup is described in detail in Figure 1.



    Figure 1. Setup for the recording of cortical activity with simultaneous electrical stimulation of the paw in the anesthetized rat.

    We used two battery-powered HS32-Ext0.5 mA headstages synchronized through the W2100 from multichannel systems (MCS). This system allowed the use of separate grounds for recording and stimulation, limiting the eStim-induced artefacts (i.e., electrical artifacts). The HS32 headstages can electrically stimulate in a bipolar setup while providing simultaneous recording through a 32-channel omnetics connector. The recording headstage was connected to a 32-lead linear silicone probe (Neuronexus). To obtain the exact time of stimulation, the stimulation headstage was connected to the paw of the animal and returned the voltage to the MSC. This system allowed a precisely synchronized stimulation and recording of events with neural response. We recorded from 2 regions (sequentially) in order to assess the laminar response of the S1 region and S1/M1 regions in the rat following paw stimulation.


  2. Controlled Cortical Injury (CCI) Surgical Procedure and eCS gel implant

    1. Prior to CCI injury, anesthetize each animal with 5% isoflurane (switched to maintenance 2-3% as soon as the incision was performed – Step B4).

    2. Inject buprenorphine (0.3 mg/ml, 0.05 ml/300g, Henry Schein) subcutaneously,

    3. Place animals with their scalp shaved and sanitized (70% Ethanol and 3% povidone-iodine) on a stereotaxic frame attached to a temperature-controlled heating pad (37°C).

    4. Perform a sagittal incision and clean the periosteum using Etch-gel (phosphoric acid etching, Henry Schein).

    5. Perform a craniotomy using a 5-mm-diameter trephine bur fitted to an electric drill.

    6. Fit a 3-mm CCI tip onto the pneumatic piston, position it in contact with the surface of the dura (fully extended position), and then retract it to adjust for an impact depth of 2 mm.

    7. Launch the 3-mm CCI tip to create an impact. A severe CCI injury is induced by programming the piston speed to 2.25 m/s with a dwell time of 250 ms, resulting in an initial 3-mm-diameter injury with a depth of 2 mm.

    8. Apply absorbable gelatin (Gel Foam®, Pfizer pharmaceutical) to the injury site, using sterile cotton swabs to remove excess blood.

    9. Remove the gelatin and completely cover the injury site with a layer of 1% sterile agarose (SeaKem®, Lonza).

    10. Suture skin flaps were together to close the wound. Apply triple antibiotic cream on the sutured skin.

    11. Optional: For the eCS group, the eCS gel was implanted 48 h post-CCI using a pre-photocrosslinked gel delivered through a 32 gauge Hamilton syringe (speed: 20 μl/10min) in the center of the CCI lesion (1 mm depth).


  3. Electrode placement surgical procedure (8 weeks post-CCI)

    1. See Structure Denomination for brain region names.

      Structure Denomination:

      1. M1: primary motor cortex

      2. RFA: rostral forelimb area, part of the M1 region for hand control

      3. CFA: caudal forelimb area, part of the M1 region for hand and leg control

      4. S1: primary sensory cortex

      5. AP/ML/DV: stereotaxic positioning relative to the bregma point (reference); AP: anterior-posterior, ML: middle-lateral, DV: dorsal-ventral

    2. From 8 to 10 weeks post-CCI, anesthetize rats that received RFA-targeted lesions using a ketamine/xylazine cocktail (100 mg/Kg) and place them on a stereotaxic frame.

    3. Following sagittal scalp incision, perform a craniotomy (Figure 2) caudally to the injury (AP: 0 mm, ML: 2 to 4mm, relatively to bregma). Using a dental drill with trephine bur (2 mm diameter), draw a square shape within the skull, from the half width of the injury site down to the bregma (AP: 0 mm), with a width of 3-4 mm, and covering the entire lateral side (from the midline to the temporal cranial muscles). Then, lift and carefully remove the bone flap with fine forceps.



      Figure 2. Position and description of the craniotomy post-lesion.


    4. Perform a durotomy to allow for the insertion of a 32-channel silicone probe (Neuronexus). Using the tip of a 1 ml insulin needle bent at 90° penetrate the dura tangentially to the brain to avoid any damage to the grey matter. Move the needle across the length of the craniotomy to tear the dura, and flip its pieces outside of the craniotomy region.

    5. Insert the silicone probe (Figure 3, Video 1) in the motor area adjacent to the RFA (position 1: CFA, AP: 0 mm, ML: 2.5 mm) or in the sensorimotor area (position 2: S1, AP: 0 mm, ML: 3.5 mm) at a depth of 2 mm from the surface of the brain. Important: since the CCI was performed on the RFA, the CFA region should remain intact. If necrosis is observed in the CFA region, chances are that the recording will yield a low number of healthy unit spiking. Necrosis in the CFA/S1 region can be identified by the presence of liquified or brownish tissue with poor consistency, missing tissue volume, or dark tissue with a mix of coagulated and non-coagulated blood.



      Figure 3. Poisitioning and setup of the recording electrode following craniotomy.

      Video 1. General setup for the terminal recording of cortical activity following paw stimulation (somatosensory evoked response). (This video was made at the University of Georgia (UGA) according to guidelines from the IACUC of UGA under protocol # A2020-06-002.)


    6. Use the probe reference (top ground electrode) as the main reference, and a screw positioned above the left cerebellum (posterior, contralateral hemisphere) as ground. The probe reference is described in the main manufacturer website: https://www.neuronexus.com/files/technicalsupportdocuments/Chronic-Wiring-Configurations.pdf (see chronic probe reference description for internal reference).


  4. Paw electrode placement procedure

    1. Use a bipolar configuration for the paw stimulation. Use close positioning of the sink and source electrode to produce a short distance electrical stimulation, which results in a very localized electrical field excitation of the tissue. The bipolar configuration is the opposite of a monopolar configuration, where the sink and source are placed far apart, with the stimulation area becoming larger and less specific as a consequence.

    2. Connect the anode and cathode of the stimulator to stainless steel needles.

    3. Insert the two electrode tips at 2 mm from each other in the ventral section of the forelimb (palm of the paw). Then, place the paw and the entire forearm on the sterile pad.


  5. Recording Procedure

    1. Digitize and record neural data at 20 kHz (unit gain) using a multichannel system (MCS) W2100 acquisition module and a wireless headstage (HS32-EXT-0.5 mA, 16 bit; Figure 4A).

    2. For spike analysis, filter the broadband electrophysiological through a real-time bandpass (300-5,000 Hz) and use a baseline pre-calculated threshold as trigger to save spike waveforms and spike event timestamps (threshold: 5 standard deviations). Do not sort for single-unit data; rather use multiunit spikes as a measure of population activity (Figure 4B, Video 2).

    3. Perform all recordings for 5 min after stabilization for 10 min.



      Figure 4. Recording setup using multichannel system software.

      Video 2. Example of recording during ketamine anesthesia with laminar electrode.


  6. Stimulation Protocols

    1. Perform all paw stimulations using a single pulse (biphasic, on phase 1 ms) with a 1 s inter-pulse interval (1 Hz). Deliver the stimulation for a total of 120 pulses (2 min). Record continuously for 5 min and set the stimulation to start after 1 min of baseline and stop 2 min before the end of the recording, to obtain a return to baseline period.

    2. Choose a stimulation intensity of 50 µA, as this induced a visible S1 response without the presence of artifacts due to electrical stimulation. Use two separate, battery-powered headstages to perform the stimulation on the paw and the recording of the electrode, allowing for independent grounding and low cross-electrical artifact formation.

    3. Co-register each stimulation triggering to the recording system to match the real delivery of stimulation, rather than the estimated onset of the stimulation.

Data analysis

  1. Analyze all data using custom-made Matlab® codes (Mathworks Inc.) and the provided Multichannel systems matlab toolbox. Upon reasonable request, the script used for analysis can be shared via email.

  2. Record data from a 32-channel linear electrode, and group recorded channels according to the laminar organization as described in Figure S11D (abe0207 manuscript) orthe graphical abstract. Use the same channel grouping for all recordings, considering that the depth (2mm from the dura) and ground position (electrode ref/ground placed in layer 1) were placed similarly (Layer 1: none; Layer 2/3: ch1-3; Layer 4: ch4-10; Layer 5A: ch11-14; Layer 5B: ch15-19; Layer 6A: ch20-29; Layer6B: ch30-32). Data analysis included:

    1. Stim-triggered histograms: use bin histograms to estimate the number of spikes elicited following the stimulation (Figure 5E; Figure S11E in Latchoumane et al., 2021).

    2. From the average multiunit waveform (Figure 6A), estimate the spike wave width and peak-to-trough values to obtain the distribution of cell type (i.e., regular spiking units showing wider widths and lower amplitudes; fast spiking units showing shorter widths and larger amplitude).

    3. Using the 5 min recording time separated into pre (1 min), STIM (2 min), and post (last 3 min), estimate the mean firing rate across channels in each layer. Compute the z-score using the mean and standard deviation of the firing rate during baseline (either a preceding recording without STIM or the pre-STIM period). This estimation can be obtained for each group (Sham, TBI, eCS) and each location (CFA and CFA/S1) (Figure 6B).

Results



Figure 5. eCS matrix implants promote sensorimotor connectivity 10 weeks post-sTBI.

A. Experimental schedule for recording session in ketamine anesthetized rats. To avoid disruption to the injury or eCS implant, use two positions for recording perilesionally for each rat. Position 1: CFA; Position 2: CFA/S1. B. Perform the recording for each position only after the probe insertion has been stabilized (top), followed by a stimulation session (2 min) with pre and post stimulation baseline recording of 1 and 2 min, respectively. Deliver 120 stimulation pulses for each position and each rat. C. The stimulation protocol used a bipolar pulse of 1 ms width (phase 1) at 1 Hz with an amplitude fixed at 50 µA for all rats. The stimulation amplitude was determined to minimize stimulation artifacts on recording while still eliciting sensorimotor response. Note: Perform stimulations and recordings using two separate wireless headstages (Multichannel Systems, W2100 HS32-Ext0.5mA) to guarantee ground and stimulation isolation from recording electrodes. Record each rat without (5 min) and with stimulation (5 min, out of which 2 min where stimulated) for the two positions CSA and CSA/S1. D. Use a 32 channel linear silicone electrode (iridium-iridium oxide, recording sites: 50 µm spacing) with a total span of approximately 1.6 mm. Perform implantation up to a depth of 2 mm from the surface of the pia. The layer position was based on the depth of the electrode and previously characterized layer distribution in the sensorimotor cortex. E. For the two recording positions, a treatment- and layer-dependent stimulation-locked response was observed. Following stimulation, a multimodal distribution typically revealed two major sharp peaks of neuronal activity (mono/di-synaptic; arrow indicating peaks 1 and 2) and later a response that revealed multi-synaptic activation of the area post-paw stimulation (arrow indicating peaks 3 and 4). Arrows indicate detected peaks of activity response, numbered in order of delay from stimulation start. F. Representative localization of the electrode positioning in a TBI rat brain, post-recording/stimulation. The arrow indicates the position of recording for position 1 and 2 shown in A. * indicates the position of the lesion. Scale bar: 300 µm.



Figure 6. Characterization of multi-unit activity in all treatment groups using linear probe recording under ketamine anesthesia.

A. Extract a multi-unit spike wave form during the resting period for each treatment group and for all recording sites. The top panel shows two representative waveforms for each treatment group, and the bottom panel shows the width vs. peak-trough length scatter plot for all detected multi-units. B. Derive a Z-score from the average population firing frequency normalized to the pre-stimulation period (1 min) for each treatment and the two recording positions CFA and CFA/S1/CFA. Note: the TBI group showed maladaptive sustained firing post stimulation for two rats out of three. Graphs show mean ± S.E.M.

Notes

Important consideration for reproducibility:

  1. Grounding issue: consider the use of battery-based separate recording and stimulation systems to reduce the chance of recording noise, electrical stimulation-induced artifacts, and other electrical interactions that might affect the recording quality.

  2. If using multi-shank silicone probes (not described in this protocol): Using probes that can span across the space and would be inserted once for the entire recording could be advantageous for this kind of recording. It would help reduce the total recording time, as well as provide simultaneous response for all layers and neighboring cortical regions involved in sensorimotor processing.

  3. Positioning of the stimulation probes: As for every silicone probe recording, use a slow speed during the insertion of the electrode to avoid sheering damages.

  4. Anesthetic depth: Perform recordings within the first 40-60 min of the ketamine-induced anesthesia to obtain the most stable data. Following a ketamine boost, the level of anesthesia might get more variable, leading to lower reproducibility. The recordings provided by the electrode are essentially local field potentials (LFP) with single neuron action potentials, which can be filtered out using 0-300Hz and 500-3000Hz bandpass filters. In particular, monitor the level of anesthesia using the LFP response of the cortical region, and include some of the delta-theta waves (0-4Hz brain waves) usually observed during the stable unconscious stage. Regularly monitor the animal response to tail and paw pinching to ensure the animal does not return to consciousness. This procedure should be performed during the recording, using the same setup as multi-unit recording.

Acknowledgments

This work was supported by an NIH (RO1NS099596) award to LK, and partially supported by a Georgia Partners in Regenerative Medicine seed grant from the Regenerative Engineering and Medicine (REM) research center to LK, and an Alliance for Regenerative Rehabilitation Research and Training (AR3T) technology development grant to LK and C-FL. Original work (Latchoumane et al., 2021), DOI: 10.1126/sciadv.abe0207.

Competing interests

There are no conflicts of interest or competing interests.

Ethics

All procedures on animals were approved by the Institutional Animal Care and Use Committee (IACUC). Protocols were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institution of Health (NIH), protocol numerb A2020-06-002 (period 2020-2023).

References

  1. Dewan, M. C., Rattani, A., Gupta, S., Baticulon, R. E., Hung, Y. C., Punchak, M., Agrawal, A., Adeleye, A. O., Shrime, M. G. and Rubiano, A. M. (2018). Estimating the global incidence of traumatic brain injury. J Neurosurg 1-18.
  2. Fernández-García, L., Marí-Buyé, N., Barios, J. A., Madurga, R., Elices, M., Pérez-Rigueiro, J., Ramos, M., Guinea, G. V. and González-Nieto, D. (2016). Safety and tolerability of silk fibroin hydrogels implanted into the mouse brain. Acta Biomater 45262-275.
  3. Hao, P., Duan, H., Hao, F., Chen, L., Sun, M., Fan, K. S., Sun, Y. E., Williams, D., Yang, Z. and Li, X. (2017). Neural repair by NT3-chitosan via enhancement of endogenous neurogenesis after adult focal aspiration brain injury. Biomaterials 14088-102.
  4. Latchoumane, C. V., Betancur, M. I., Simchick, G. A., Sun, M. K., Forghani, R., Lenear, C. E., Ahmed, A., Mohankumar, R., Balaji, N. and Mason, H. D. (2021). Engineered glycomaterial implants orchestrate large-scale functional repair of brain tissue chronically after severe traumatic brain injury. Sci Adv 7(10): eabe0207.
  5. Tan, H. X., Borgo, M. P. D., Aguilar, M. I., Forsythe, J. S., Taylor, J. M. and Crack, P. J. (2020). The use of bioactive matrices in regenerative therapies for traumatic brain injury. Acta Biomater 1021-12.
  6. Yang, Z., Zhang, A., Duan, H., Zhang, S., Hao, P., Ye, K., Sun, Y. E. and Li, X. (2015). NT3-chitosan elicits robust endogenous neurogenesis to enable functional recovery after spinal cord injury. Proc Natl Acad Sci U S A 112(43): 13354-13359.

简介

[摘要]严重创伤性脑损伤 (sTBI) 幸存者经历永久性功能障碍由于显着的体积损失和大脑再生能力差。硫酸软骨素糖胺聚糖 (CS-GAG) 是生长因子信号传导和神经干细胞的关键调节剂大脑中的稳态。在本协议中,我们描述了如何进行录音以量化植入工程 CS-GAG 水凝胶 (eCS) 对大脑的神经保护和再生作用组织。该实验在三种条件下对大鼠进行:健康无损伤(Sham),前肢前肢区域 (RFA) 上的受控皮质撞击 (CCI) 损伤,以及带有 eCS 植入物的 CCI-RFA。该协议描述了用于执行开颅手术的程序,皮质的定位记录电极,刺激电极(对侧爪)的定位,以及记录程序。此外,还提供了准确电气设置的说明。该协议详细说明记录受伤动物的大脑,同时保留大部分未受伤的组织完整,多单元反应的病灶内和层流记录的其他考虑因素。

[背景]创伤性脑损伤 (TBI) 是一个常见且日益普遍的问题,它影响着迄今为止,全球约有 6900 万人尚未获得有效治疗(Dewan等人,2018 年)。鉴于二级神经保护策略的失败,例如减压开颅术或紧血压调节,在改善不良功能结果方面,越来越多的注意力转向通过生物材料植入物重建大脑中受损的神经元回路,有或没有并发细胞移植(Tan等,2020)。除了提供注入任何一种的能力几种天然或合成的生物材料成一个损伤病灶,尚不清楚这些有什么功能影响植入物具有恢复天然神经元回路以及更高阶的认知和运动结果。在我们的手稿中(Latchoumane等人,2021 年,DOI:10.1126/sciadv.abe0207),我们给老鼠植入了工程化硫酸软骨素糖胺聚糖 (eCS) 作为组织丢失的潜在治疗方法TBI 后运动功能丧失。评估促进的生理恢复eCS 植入物,我们记录了响应爪子电刺激的层流皮质活动在麻醉的大鼠中。该领域以前的工作在300 上使用了离体平面多电极阵列用于评估场兴奋性突触后电位 (fEPSPs) 后生物材料植入的 µm 脑切片(Yang等人,2015 年;Hao等人,2017 年)。其他实验室已经进行了稳态诱发电位(SSEP)在小鼠大脑中长期植入电极的录音,以测量大脑反应植入物(Fernández-García等人,2016 年)。我们提出了一个简单的协议,可以在一个单一的程序,使用多通道系统记录和刺激设置来评估TBI 后植入 eCS 的动物的感觉运动整合。我们的协议证明了危险地记录以评估生物材料整合的可行性和可重复性,周围组织上的 eCS,以及植入物内部和周围的活神经元增殖程度。

关键字:线性硅胶探头, 尾前肢区, 爪子刺激, 层状皮质记录, 多单元感官反应

材料和试剂

1.缝合4-0 Ethicon Absorbable plus抗菌剂(Vicryl,目录号:109162

2. 自攻螺钉(18-8 不锈钢开槽平头螺钉,M0.8 × 0.2 mm 螺纹,2毫米长;McMastercarr,目录号:91430A143

3. 无菌棉签,棉签头 6"/Sterile 100/box (Dynarex)

4. 刺激针,无菌不锈钢针 24 号(BDMicrolance,目录号:1730738)

5. 动物:Sprague-Dawley 大鼠,雄性,年龄(7-10 周)(Charles River,目录号:400

6. 32 通道线性探头(NeuronexusA1x32-6mm-50-177-CM3215 µm 厚度,Gen4,批次# P994)

7. 氯胺酮 100 mg/mlCoventrus,目录号:056347361-4

8. 甲苯噻嗪鸡尾酒,100mg/mlSigma-Aldrich,目录号:7361-61-7

9.异氟烷(Coventrus,目录号:029405

10.丁丙诺啡,0.03 mg/mlCoventrus,目录号:059122

11. Marcaine0.5%Coventrus,目录号:054893

12. 聚维酮碘,10% 局部溶液(CVS,目录号:59779-085

13. Etch-Gel,磷酸 40%DMG,目录号:61901

14. Gel Foam ®(辉瑞,目录号:115631

15. SeaKem ®琼脂糖(Lonza,目录号:50004

16. 三重抗生素,0.9 克袋装(25 克拉盒)(Safetec,目录号:53205

17. 氯胺酮/赛拉嗪鸡尾酒(见食谱)

氯胺酮/甲苯噻嗪鸡尾酒:

对于 1 毫升溶液:0.9 毫升氯胺酮 (100 毫克/毫升) + 0.1 毫升甲苯噻嗪 (100 毫克/毫升)

● KX 大鼠鸡尾酒 0.1 毫升/100 克大鼠重量。IP(氯胺酮:90 毫克/公斤,甲苯噻嗪:9.0 毫克/公斤)


设备

1、多通道采集系统(MCS,无线录音,型号:W2100

2、多通道记录刺激探头(MCSheadstage,型号:HS32-EXT0.5mA

3. 戴尔 PC i7RAM8GoSSD500 GoPremium Silicone

4. Kopf 带操纵臂的啮齿动物立体定向框架

5.电子面包板(半尺寸面包板;63adafruit.com

6. 电子电缆(包覆公-公跳线,200 mm × 40Adafruit.com,目录编号:4482

7. MCS 同轴 TTL 电缆(C-BNC-Lemo1m;多通道系统附件)

8.电动剃须刀(飞利浦,Norelco oneblade QP2520/90

9. 电钻和环钻(微电机世阳,H102S

10. CCI 尖端,直径 3 毫米(定制)

11. CCI冲击机(UGA车间定制)


软件

1. 收购软件:控制系统实验者(多通道系统,https://www.multichannelsystems.com/ )

2.系统配置:控制系统配置文件(多通道系统,https://www.multichannelsystems.com/ )

3. 数据分析:MATLAB+控制系统工具箱(多通道系统,https://www.multichannelsystems.com/ )

4. Matlab R2019b (Mathworks Inc., mathworks.com )


程序

A.系统设置

系统设置被详细描述在图1中描述。

我们使用了两个通过 W2100 同步的电池供电的 HS32-Ext0.5 mA 探头来自多通道系统(MCS)。该系统允许使用单独的理由记录和刺激,限制了 eStim 诱发的伪影(即电伪影)。这HS32的探头可在一个双极电设置刺激而提供同时通过一个 32 通道的记忆连接器进行录音。录音探头已连接到 32 导联线性硅胶探头 (Neuronexus)。为了获得准确的刺激时间,刺激探头连接到动物的爪子并将电压返回到MSC。该系统允许的事件有一个精确的同步刺激和记录神经反应。我们从 2 个区域(按顺序)记录以评估层流爪刺激后大鼠 S1 区和 S1/M1 区的反应。


B. 受控皮质损伤 (CCI) 手术程序和 eCS 凝胶植入物

1. CCI 损伤之前,用 5% 异氟醚麻醉每只动物(切换到维持 2-3%一旦进行了切口——步骤 B4)

2. 皮下注射丁丙诺啡 (0.3 mg/ml, 0.05 ml/300g, Henry Schein)

3. 将头皮剃光和消毒(70% 乙醇和 3% 聚维酮碘)的动物放在连接到温控加热垫 (37°C) 的立体定位框架。

4. 进行矢状切口并使用蚀刻凝胶(磷酸蚀刻,亨利施恩)。

5. 使用安装在电钻上的 5 毫米直径环钻进行开颅手术。

6. 3 毫米 CCI 尖端安装到气动活塞上,使其与硬脑膜表面接触(完全伸展位置),然后缩回以调整 2 毫米的冲击深度。

7. 启动 3 毫米 CCI 尖端以产生冲击。编程诱发了严重的 CCI 损伤活塞速度达到 2.25 m/s,停留时间为 250 ms,导致初始直径为 3 mm深度为 2 毫米的损伤。

8.在受伤部位涂抹可吸收明胶(Gel Foam ®,辉瑞制药),使用无菌棉签去除多余的血液。

9. 去除明胶,用一层 1% 无菌琼脂糖完全覆盖损伤部位(SeaKem ®Lonza)。

10.缝合皮瓣在一起以闭合伤口。在缝合采用三层抗生素霜皮肤。

11.可选:对于 eCS 组,eCS 凝胶在 CCI 48 小时使用预光交联凝胶通过 32 Hamilton 注射器(速度:20 μl/10 分钟)在CCI 病变的中心(1 毫米深)。


C. 电极放置手术程序(CCI 8 周)

1. 大脑区域名称见结构命名。

结构名称:

●M1:初级运动皮质

●RFA:喙前肢区域,用于手动控制的M1区域的一部分。

● CFA:前肢尾部区域,用于手和腿控制的 M1 区域的一部分

● S1:初级感觉皮层

● AP/ML/DV:相对于前囟点(参考)的立体定位;AP:前-后部,ML:中外侧,DV:背腹侧

2. CCI 8 10 周,使用麻醉剂麻醉接受 RFA 靶向病变的大鼠氯胺酮/赛拉嗪混合物(100毫克/千克),并代替立体定位框架上。

3. 在矢状头皮切口后,在损伤尾部进行开颅手术(图 2)(AP0mmML2 4mm,相对于 bregma)。使用带环钻(直径 2 毫米)的牙钻,在头骨内画一个正方形,从受伤部位的一半宽度到前囟(AP0毫米),具有3-4毫米的宽度,并且覆盖整个横向侧(从中线到颞颅肌)。然后,用细镊子提起并小心取出骨瓣。

4. 进行硬脑膜切开术以插入 32 通道硅胶探头 (Neuronexus)使用将 1 ml 胰岛素针头弯曲 90° 的尖端切向于大脑,以避免对灰质的任何损害。将针移过开颅手术的长度以撕裂硬脑膜,并在开颅区域外翻转其碎片。

5.将硅酮探针(图3,视频1)在运动区邻近于所述RFA(位置1CFAAP0 毫米,ML2.5 毫米)或在感觉运动区域(位置 2S1AP0 毫米,ML3.5毫米)在距离大脑表面 2 毫米的深度处。重要:由于执行了 CCIRFA,终审法院地区应该保持不变。如果坏死的CFA区域观察,记录可能会产生少量的健康单位尖峰。坏死CFA/S1 区域可以通过液化或褐色组织的存在来识别稠度、组织体积缺失或黑色组织混合凝固和未凝固血液。

6. 使用探针参考(顶部接地电极)作为主要参考,并用螺钉定位左小脑上方(后侧,对侧半球)作为地面。探头参考是描述在这主要的制造商网站:

https://www.neuronexus.com/files/technicalsupportdocuments/Chronic-Wiring- Configurations.pdf (有关内部参考,请参阅长期探针参考描述)。


D. 爪电极放置程序

1. 使用双极配置进行爪子刺激。使用接收器和源的紧密定位电极产生短距离电刺激,从而导致非常局部化组织的电场激发。双极配置与单极配置相反配置,其中接收器和源放置得很远,带有刺激区域结果变得更大,更不具体。

2. 将刺激器的阳极和阴极连接到不锈钢针上。

3. 将两个电极尖端相距 2 mm 插入前肢(手掌)的腹侧部分爪子)。然后,将爪子和整个前臂放在无菌垫上。


E. 记录程序

1. 使用多通道系统 (MCS) W2100 20 kHz(单位增益)数字化和记录神经数据采集模块和无线探头(HS32-EXT-0.5 mA16 位;图 4A)。

2. 对于尖峰分析,通过实时带通 (300-5,000 Hz)并使用基线预先计算的阈值作为触发器来保存尖峰波形和尖峰事件时间戳(阈值:5 个标准差)。不要对单个单元数据进行排序;而是使用多单元峰值作为人口活动的衡量标准(图 4B,视频 2)。

3. 稳定 10 分钟后执行所有记录 5 分钟。


F. 刺激方案

1. 使用单脉冲(双相,相位 1 毫秒)执行所有爪子刺激,间隔为 1 秒。脉冲间隔 (1 Hz)。提供总共 120 个脉冲(2 分钟)的刺激。记录连续 5 分钟,并将刺激设置为在基线 1 分钟后开始并停止 2 分钟在记录结束之前,获得返回到基线期。

2. 选择 50 µA 的刺激强度,因为这会在没有由于电刺激而存在伪影。使用两个独立的电池供电Headstages 对爪子进行刺激并记录电极,允许用于独立接地和低交叉电气伪影形成。

3. 将每个刺激触发共同注册到记录系统以匹配实际交付刺激,而不是刺激的估计开始时间。


数据分析

1. 使用定制的 Matlab ®代码(Mathworks Inc.)和提供的数据分析所有数据多通道系统 matlab 工具箱。根据合理要求,用于分析的脚本可以通过电子邮件共享。

2. 记录一个32通道线性电极的数据,并根据记录的通道分组如图 S11Dabe0207 手稿)或图形所示的层状组织抽象的。对所有录音使用相同的通道分组,考虑到深度(2mm从硬脑膜)和接地位置(电极参考/接地放置在第 1 层)的位置类似(第 1 层:无;第 2/3 层:ch1-3;第 4 层:ch4-10;第 5A 层:ch11-14;第 5B 层:ch15-19;第6Ach20-29;第 6B 层:ch30-32)。数据分析包括:

 a.  刺激触发的直方图:使用 bin 直方图来估计引发的尖峰数量在刺激之后(图 5E;图 S11E 来自 Latchoumane等人2021 年)。

 b. 从平均多单元波形(图 6A),估计尖峰波宽度和峰值 -到谷值以获得细胞类型的分布(,规则的尖峰单位显示更宽的宽度和更低的振幅;显示较短宽度和较大宽度的快速尖峰单元振幅)。

 c. 使用分为前(1 分钟)、STIM2 分钟)和后(最后 3 分钟)的 5 分钟录音时间min),估计每层中跨通道的平均发射率。计算 z 分数使用基线期间放电率的平均值和标准差(前一个没有 STIM 或前 STIM 期间的记录)。这个估计可以为每个组 (ShamTBIeCS) 和每个位置 (CFA CFA/S1) ( 6B)

结果


笔记

再现性的重要考虑因素:

1. 接地问题:考虑使用基于电池的单独记录和刺激系统减少记录噪音、电刺激引起的伪影和其他可能影响记录质量的电相互作用。

2. 如果使用多柄硅胶探头(本协议中未描述):使用可以跨越跨越空间,并在整个录音中插入一次可能是有利的对于这种录音。这将有助于减少总录音时间,并提供参与感觉运动的所有层和相邻皮层区域的同时响应加工。

3.刺激探头的定位:对于每一个硅胶探头录音,使用慢速在插入电极的过程中,以避免剪切损坏。

4. 麻醉深度:在氯胺酮诱导的前 40-60 分钟内进行录音麻醉以获得最稳定的数据。氯胺酮加强后,麻醉水平可能会变得更加可变,从而导致较低的重现性。提供的录音电极本质上是具有单个神经元动作电位的局部场电位 (LFP),它可以使用 0-300Hz 500-3000Hz 带通滤波器滤除。特别是,监测使用皮层区域的 LFP 反应确定麻醉水平,并包括一些 delta-Theta 波(0-4Hz 脑电波)通常在稳定的无意识阶段观察到。定期监测动物对捏尾巴和爪子的反应,以确保动物不会回归意识。此过程应在记录期间执行,使用与多单元录音相同的设置。


致谢

这项工作得到了 LK NIH (RO1NS099596) 奖的支持,并部分得到了一个佐治亚州再生工程和再生医学种子基金的合作伙伴LK 的医学 (REM) 研究中心和再生康复研究联盟和培训 (AR3T) 技术开发补助金给 LK C-FL。原作 (Latchoumane等。, 2021), DOI: 10.1126/sciadv.abe0207


利益争夺

不存在利益冲突或竞争利益。


伦理

动物的所有程序都得到了机构动物护理和使用委员会的批准(IACUC)。方案是根据护理和使用指南进行的美国国立卫生研究院 (NIH) 发布的实验动物,协议编号 A2020-06-0022020-2023 年期间)。


参考

1. Dewan, MC, Rattani, A., Gupta, S., Baticulon, RE, Hung, YC, Punchak, M., Agrawal, A.,AdeleyeAOShrimeMGRubianoAM等。(2018)估计全球发病率创伤性脑损伤。神经外科杂志1-18

2. Fernández-García, L., Marí-Buyé, N., Barios, JA, Madurga, R., Elices, M., Pérez-Rigueiro, J.,Ramos, M.Guinea, GV González-Nieto, D. (2016)丝素蛋白的安全性和耐受性水凝胶植入小鼠大脑。 生物材料学报45262-275

3. Hao, P., Duan, H., Hao, F., Chen, L., Sun, M., Fan, KS, Sun, YE, Williams, D., Yang, Z. Li, X. (2017) NT3-壳聚糖通过增强内源性神经发生进行神经修复成人局灶性吸入脑损伤后。生物材料14088-102

4. Latchoumane, CV, Betancur, MI, Simchick, GA, Sun, MK, Forghani, R., Lenear, CE,Ahmed, A.Mohankumar, R.Balaji, N.Mason, HD(2021)工程糖材料植入物在严重后长期协调脑组织的大规模功能修复创伤性脑损伤。Sci Adv 710):eabe0207

5. Tan, HX, Borgo, MPD, Aguilar, MI, Forsythe, JS, Taylor, JM Crack, PJ (2020)生物活性基质在创伤性脑损伤再生疗法中的应用。生物材料学报1021-12

6. Yang, Z., Zhang, A., Duan, H., Zhang, S., Hao, P., Ye, K., Sun, YE Li, X. (2015) NT3-壳聚糖引发强大的内源性神经发生,使脊髓后功能恢复成为可能受伤。 Proc Natl Acad Sci USA 112(43): 13354-13359

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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Latchoumane, C. V., Forghani, R. and Karumbaiah, L. (2021). Cortical Laminar Recording of Multi-unit Response to Distal Forelimb Electrical Stimulation in Rats. Bio-protocol 11(22): e4153. DOI: 10.21769/BioProtoc.4153.
  2. Latchoumane, C. V., Betancur, M. I., Simchick, G. A., Sun, M. K., Forghani, R., Lenear, C. E., Ahmed, A., Mohankumar, R., Balaji, N. and Mason, H. D. (2021). Engineered glycomaterial implants orchestrate large-scale functional repair of brain tissue chronically after severe traumatic brain injury. Sci Adv 7(10): eabe0207.
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