Name: establishment of acute pontine infarction in rats by electrical stimulation
Uploaded: 2020-08-27T13:00:00
Description: Watch this Scientific Journal Video about Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation at JoVE.com

This study was financially supported by the National Science Foundation of China (81471181 and 81870933) to Y. Jiang and the National Science Foundation of China (No. 81601011), Natural Science Foundation of Jiangsu Province (No. BK20160345) to J. Zhu and by the Scientific Program of Guangzhou Municipal Health Commission (20191A011083) to Z. Qiu.

The protocol was reviewed and approved by the Institution Animal Care and Use Committee of The Second Affiliated Hospital of Guangzhou Medical University, an institution accredited by AAALACi. The rats were provided by the Animal Center of Southern Medical University.
1. Animal
<ol>
	<li>Use adult male Sprague-Dawley rats weighing 250 &#177; 10 g.</li>
	<li>Upon transport, house the rats for at least 1 week before surgery under controlled environmental conditions with an ambient temperature ...

<ol>
	<li>Zhu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+local+inflammation+contributes+to+the+neuroprotective+effect+of+ginsenoside+Rb1+in+rats+with+cerebral+ischemia.">Suppression of local inflammation contributes to the neuroprotective effect of ginsenoside Rb1 in rats with cerebral ischemia.</a> Neuroscience. 202, 342-351 (2012).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-1906,+a+Novel+Regulator+of+Toll-Like+Receptor+4,+Ameliorates+Ischemic+Injury+after+Experimental+Stroke+in+Mice.">MicroRNA-1906, a Novel Regulator of Toll-Like Receptor 4, Ameliorates Ischemic Injury after Experimental Stroke in Mice.</a> Journal of Neuroscience. 37, 10498-10515 (2017).</li><li>McBride, D. W., Zhang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+Stroke+Animal+Models:+the+Permanent+MCAO+Model+Should+Be+the+Primary+Model,+Not+Transient+MCAO.">Precision Stroke Animal Models: the Permanent MCAO Model Should Be the Primary Model, Not Transient MCAO.</a> Translational Stroke Research. , (2017).</li><li>Liu, F., McCullough, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Middle+cerebral+artery+occlusion+model+in+rodents:+methods+and+potential+pitfalls.">Middle cerebral artery occlusion model in rodents: methods and potential pitfalls.</a> Journal of Biomedicine &amp; Biotechnology. 2011, 464701 (2011).</li><li>Jiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+with+less+damage:+intranasal+delivery+of+tetracycline-inducible+replication-defective+herpes+simplex+virus+type-1+vector+to+brain.">A new approach with less damage: intranasal delivery of tetracycline-inducible replication-defective herpes simplex virus type-1 vector to brain.</a> Neuroscience. 201, 96-104 (2012).</li><li>Lopez, M. S., Vemuganti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Transient+Focal+Ischemic+Stroke+in+Rodents+by+Intraluminal+Filament+Method+of+Middle+Cerebral+Artery+Occlusion.">Modeling Transient Focal Ischemic Stroke in Rodents by Intraluminal Filament Method of Middle Cerebral Artery Occlusion.</a> Methods in Molecular Biology. 1717, 101-113 (2018).</li><li>Pais-Roldan, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+assessment+of+recovery+from+coma+in+a+rat+model+of+diffuse+brainstem+tegmentum+injury.">Multimodal assessment of recovery from coma in a rat model of diffuse brainstem tegmentum injury.</a> NeuroImage. 189, 615-630 (2019).</li><li>Merwick, A., Werring, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+circulation+ischaemic+stroke.">Posterior circulation ischaemic stroke.</a> The British Medical Journal. 348, 3175 (2014).</li><li>Nogueira, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombectomy+6+to+24+Hours+after+Stroke+with+a+Mismatch+between+Deficit+and+Infarct.">Thrombectomy 6 to 24 Hours after Stroke with a Mismatch between Deficit and Infarct.</a> The New England Journal of Medicine. 378, 11-21 (2018).</li><li>Wilkinson, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+recanalization+of+basilar+artery+occlusion+in+a+previously+healthy+17-month-old+child.">Late recanalization of basilar artery occlusion in a previously healthy 17-month-old child.</a> Journal of Neurointerventional Surgery. 10, 17 (2018).</li><li>Huang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke+Subtypes+and+Topographic+Locations+Associated+with+Neurological+Deterioration+in+Acute+Isolated+Pontine+Infarction.">Stroke Subtypes and Topographic Locations Associated with Neurological Deterioration in Acute Isolated Pontine Infarction.</a> Journal of Stroke and Cerebrovascular Diseases: The Official Journal of National Stroke Association. 25, 206-213 (2016).</li><li>Jiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-stent+restenosis+after+vertebral+artery+stenting.">In-stent restenosis after vertebral artery stenting.</a> International Journal of Cardiology. 187, 430-433 (2015).</li><li>Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographic+location+of+unisolated+pontine+infarction.">Topographic location of unisolated pontine infarction.</a> BMC Neurology. 19, 186 (2019).</li><li>Banerjee, G., Stone, S. P., Werring, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+circulation+ischaemic+stroke.">Posterior circulation ischaemic stroke.</a> The British Medical Journal. 361, 1185 (2018).</li><li>Wojak, J. C., DeCrescito, V., Young, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basilar+artery+occlusion+in+rats.">Basilar artery occlusion in rats.</a> Stroke: A Journal of Cerebral Circulation. 22, 247-252 (1991).</li><li>Namioka, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+infusion+of+mesenchymal+stem+cells+for+protection+against+brainstem+infarction+in+a+persistent+basilar+artery+occlusion+model+in+the+adult+rat.">Intravenous infusion of mesenchymal stem cells for protection against brainstem infarction in a persistent basilar artery occlusion model in the adult rat.</a> Journal of Neurosurgery. , 1-9 (2018).</li><li>Jiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+brain-derived+neurotrophic+factor+protects+brain+from+ischemic+insult+via+modulating+local+inflammation+in+rats.">Intranasal brain-derived neurotrophic factor protects brain from ischemic insult via modulating local inflammation in rats.</a> Neuroscience. 172, 398-405 (2011).</li><li>Schaar, K. L., Brenneman, M. M., Savitz, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessments+in+the+rodent+stroke+model.">Functional assessments in the rodent stroke model.</a> Experiments in Translational and Stroke. 2, 13 (2010).</li><li>Wu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keep+warm+and+get+success:+The+role+of+postischemic+temperature+in+the+mouse+middle+cerebral+artery+occlusion+model.">Keep warm and get success: The role of postischemic temperature in the mouse middle cerebral artery occlusion model.</a> Brain Research Bulletin. 101, 12-17 (2014).</li><li>Wen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+behavioural+tests+for+the+prediction+of+outcomes+in+mouse+models+of+focal+middle+cerebral+artery+occlusion.">Optimization of behavioural tests for the prediction of outcomes in mouse models of focal middle cerebral artery occlusion.</a> Brain Research. 1665, 88-94 (2017).</li></ol>

The present study provides a protocol for generating an acute pontine infarction rat model. This model can be used for research on prognosis and rehabilitation (including post-stroke chronic pain) in pontine stroke patients.
There are several strengths of this method. First, it provides a rat model of acute pontine infarction for future studies. As mentioned above, pontine infarction is a common stroke subtype that has received less attention. A major shortcoming of stroke research has been th...

Since the 1980s, the middle cerebral artery occlusion (MCAO) model induced by silicone filaments has been widely used in basic stroke research1. Other methods (i.e., suturing of one branch of the MCA2 and photochemically induced focal infarction) have also been used. These models have been termed MCA-based stroke models and have greatly contributed to investigations of the pathophysiological mechanisms underlying stroke and potential therapeutics. Although there are limitations of these experimental models3,4, these methods have been used many labs5,6. MCA-based stroke models represent a stroke in the anterior circulation; however, few reports have investigated models mimicking stroke in the posterior circulation7.
There are significant differences among the etiology, mechanisms, clinical manifestation, and prognosis between anterior and posterior circulation strokes8. Therefore, the results derived from anterior circulation stroke models cannot be applied to posterior circulation stroke. For example, the reperfusion time window for anterior circulation has been extended to 6 h, with a small portion of studies extending to 24 h based on imaging findings9. However, the time window for posterior circulation may be longer than 24 h, according to previous reports10 and our own clinical experiences. This elongated reperfusion time window must be further studied and confirmed in experimental models.
Regarding posterior circulation strokes, pontine infarction is the most common subtype, accounting for 7% of all ischemic stroke cases11,12. According to infarction topography, pontine infarctions are divided into isolated and non-isolated pontine infarctions13. Isolated pontine infarctions are categorized into three types based on the underlying mechanisms: large artery disease (LAD), basilar artery branch disease (BABD), and small artery disease (SAD). Knowledge of the mechanisms, manifestation, and prognosis of pontine infarction has been derived from clinical investigations of cases14. However, a rodent model mimicking pontine infarction has been less investigated.
In previous studies, diffuse brainstem tegmentum injury involving the pons has been explored7. One group attempted to create a pontine infarction model via ligation of the basilar artery (BA)15. Another group used a 10-0 nylon monofilament suture to selectively ligate four points of the proximal BA selectively16. This model mimics LAD, but most pontine infarctions result from BABD and SAD. In addition, selective ligation of the BA is a complicated surgery and has a high death rate.
Provided here is a detailed protocol for an easy-to-perform, easily reproduced, and successful rat model of acute pontine infarction by electrical stimulation.

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	<tbody>
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			<td height="42" style="height:42px;width:215px;">4-0 sucture</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td style="width:167px;">Surgical instruments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Adhesive tape</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Animal anesthesia system</td>
			<td style="width:121px;">RWD</td>
			<td style="width:161px;"></td>
			<td>Wear mask when using the system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Bone cement</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Cured clamp</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">General tissue scissors</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">IndoPhors</td>
			<td style="width:121px;">Guoyao of China</td>
			<td style="width:161px;"></td>
			<td>Sterilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Isoflurane</td>
			<td style="width:121px;">RWD</td>
			<td align="right" style="width:161px;">217181101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Lesion Making Device</td>
			<td style="width:121px;">Shanghai Yuyan</td>
			<td style="width:161px;"></td>
			<td>Making a lesion</td>
		</tr>
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			<td height="21" style="height:21px;width:215px;">MRI system</td>
			<td style="width:121px;">Bruker Biospin</td>
			<td style="width:161px;"></td>
			<td>Confirmation of infarction in vivo</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Needle holder</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Penicilin</td>
			<td style="width:121px;">Guoyao of China</td>
			<td style="width:161px;"></td>
			<td>Infection Prevention</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Probe</td>
			<td style="width:121px;">Anke</td>
			<td style="width:161px;"></td>
			<td>Need some modification</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Q-tips</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Shearing scissors</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Stereotaxic apparatus</td>
			<td style="width:121px;">RWD</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Suture needle</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Tissue holding forcepts</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">TTC</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">BCBW5177</td>
			<td>For infarction confirmation in vitro</td>
		</tr>
	</tbody>
</table>

establishment of acute pontine infarction in rats by electrical stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Six animals were subjected to the surgery protocol described above. The control group as shown in the Figure 4 consisted of six rats. The brain slices shown in the Figure 4 were derived from one rat per group.
The MRI scanning showed that the infarction was located in the basis of the pons (Figure 4A). Since the probe was injected 2 mm to the left of the midline, the infarction was located laterally. This...

Watch this Scientific Journal Video about Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation at JoVE.com

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

We provide a protocol to establish infarction in a pons of rat. This pontine infarction rat model was easily performed, easily reproduced, and highly successful model. The electrical stimulation could be used to make infarction in any other brain area like thalamus.People who want to perform this model should learn the pontine autonomy before the experiment. Before the surgery, weigh the rats and assess their neurological performance according to the behavioral tests as described in the accompanying text. Preheat the heating pad to 37 degrees Celsius immediately before anesthesia and attach the skull drill to the holder on the stereotaxic frame.Confirm a lack of response to pedal reflex in the first anesthetized rat and mount the rat onto the stereotaxic frame in the prone position on the heating pad. Position the ear bars above the ear canal to secure the head, taking care that the skull is kept horizontal to avoid any skewing of the injection. Apply ointment to the animal's eyes and use a micro shaver to remove the hair from the skull.Use a marker to draw a three centimeter line along the midline of the skull from the line of the bilateral lateral canthus to 0.5 centimeters behind the posterior fontanelle and use a scalpel to make a midline incision along the marked line. Use a cotton swab to remove any blood and place a piece of surgical tape on each side of the skin flap to expose the skull. Use a new swab soaked with 0.9%sodium chloride to gently remove any connective tissue from the skull and locate the bregma.Use a fine tip marker pen to indicate the central point of the bregma as the origin point, place the drill at six millimeters anterior posterior, two millimeters medial lateral. Use the drill to carefully perform a one millimeter diameter craniotomy. Next, replaced the drill with a 22 gauge probe with an insulated sheath with the tip placed two millimeters above the proximal end of the sheath.Insert the sheets seven millimeters into the brain and advance the probe along the sheath until the top of the probe is nine millimeters below the surface of the brain. Connect the electrodes to an electrical stimulator and connect the anode to the probe. Then connect to the cathode to the ear of the rat.Next, turn on the electrical stimulator and set the single pulse width to 4, 050 milliseconds, the voltage to 50 volts and the current to four milliamps. Leave the probe in position for five minutes after stimulation before removing the device from the brain. Cover the craniotomy with bone cement and allow the cement to dry before using 4.0 polyamide suture filaments to close the wound.After three or four stitches, tie 2-1-1 standard surgical knots. Then intraperitoneally inject the rat with 250 microliters of penicillin and monitor the rat every 15 minutes until fully awake before returning the animal to its cage. In this analysis, six animals were subjected to the surgery as demonstrated and six animals were treated as controls.The brain slices in these images were derived from one rat per group. MRI scanning revealed that the infarction was located in the basis of the pons mimicking anterolateral infarctions in human patients. Because an insulated sheath was used, the infarction was not observed in the cortex, cerebellum, or midbrain.Diffusion weighted images also revealed the acute pontine infarction. TTC staining was used to confirm the infarction 24 hours post-surgery. As expected, the infarction volume was significantly higher in the surgery group compared to the control group.Behavioral scores were measured before and after surgery. Compared to the control group, rats with a pontine infarction circled to the left. Significant differences were observed in the Longa, Berderson, limb placement, beam balance, and adhesive removal somatosensory test scores between rants with pontine infarction and control group rats.Any specific brain area are going to be destroyed by the electrical stimulation.

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Research

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Neuroscience

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala &#8211; peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 &#176;C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases.

Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, and neuroinflammation is often observed as a co-occurrence. Here, we describe a sub-acute CMHs rat model induced by lipopolysaccharide (LPS) injection, as well as a method for detecting CMHs. Systemic LPS injection is relatively simple, economical, and cost-effective. One major advantage of LPS injection is its stability to induce inflammation. CMHs caused by LPS injection could be detected by gross observation, hematoxylin and eosin (HE) staining, Perl&#39;s Prussian staining, Evans blue (EB) double-labeling, and magnetic resonance imaging-susceptibility weighted imaging (MRI-SWI) technology. Finally, other methods of developing CMHs animal models, including their advantages and/or disadvantages, are also discussed in this report.

We present a protocol to induce and detect CMHs caused by LPS injection in Sprague-Dawley rats, which may be utilized in future research investigations on the pathogenesis of CMHs.

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

Massive Pontine Hemorrhage by Dual Injection of Autologous Blood

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred microliters of autologous blood was taken from the tail vein and stereotaxically injected into the pons. The injection process was divided into 2 steps: First, 10 &#181;L of blood was injected into a specific location, anteroposterior position (AP) -9.0 mm; lateral (Lat) 0 mm; vertical (Vert) -9.2 mm, followed by a second injection of the residual blood located at AP -9.0 mm; Lat 0 mm; Vert -9.0 mm with a 20-minute interval. The balance beam test, limb placement test, and the modified Voestch neuroscore were used to evaluate neurological function. Magnetic Resonance Imaging (MRI) was used to assess the volume of hemorrhage in vivo. The symptoms of this model were in line with patients with massive pontine hemorrhage.

We present a protocol to establish a massive pontine hemorrhage model in a rat via dual injection of autologous blood.