Name: rating l-dopa-induced dyskinesias in the unilaterally 6-ohda-lesioned rat model of parkinson's disease
Uploaded: 2021-10-04T14:30:00
Description: Watch this Scientific Journal Video about Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease at JoVE.com

This work was supported by S&#227;o Paulo Research Foundation (FAPESP, grant 2017/00003-0). We are grateful for the Coordination for the Improvement of Higher Education Personnel (CAPES). We thank Dr. Anthony R. West, Dr. Heinz Steiner, and Dr. Kuei Y. Tseng for support and mentoring.

All experiments were performed in accordance with The Ethics Committee of the Faculty of Philosophy, Sciences, and Letters of Ribeir&#227;o Preto (CEUA/FFCLRP 18.5.35.59.5).
1.&#160;6-OHDA lesion
<ol>
	<li>Use Sprague-Dawley male rats weighing 200-250 g at the beginning of the experiments (6 weeks). House the animals (2-3 per cage) under standard laboratory conditions (12:12 h light/dark cycle, lights on at 06:00 h, temperature-controlled facilities (22-24 &#176;C), with foo...

This protocol demonstrates how to induce and analyze AIMs in the rat model of PD induced by unilateral microinjection of 6-OHDA in the MFB. Chronic daily administration of low doses of L-DOPA (5 mg/kg, combined with 12.5 mg/kg of benserazide) produced the development of AIMs over the 3 weeks of treatment. Temporal analysis revealed a significant increase of AIMs, and the peak-dose dyskinesia is observed between 30 and 90 min after L-DOPA administration. AIMs are repetitive and purposeless movements affecting axial, limb,...

The dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) represents the most effective treatment for the motor symptoms of Parkinson&#39;s disease (PD)1. L-DOPA therapy may ameliorate motor symptoms associated with PD but loses effectiveness with time. Motor fluctuations such as &#34;wearing-off fluctuation&#34; or &#34;end-of-dose deterioration&#34; manifest clinically as a shortened duration of the effect of single L-DOPA doses2. In other cases, clinical manifestations consist of slow twisting movements and abnormal postures (dystonia)3 and occur when dopamine levels are low (off-period dystonia)4. On the other hand, L-DOPA-induced dyskinesias (LIDs) appear when dopamine levels in the plasma and the brain are high5.
LIDs produce debilitating side effects that include motor complications such as choreiform, dystonic, and ballistic6 movements. Once established, LIDs occur after every L-DOPA administration. Motor complications occur in 40%-50% of PD patients undergoing L-DOPA therapy for 5 years, and the incidence increases over the years7. Although the pathophysiological mechanisms involved in the development of LIDs in PD patients are not yet fully elucidated, the extent of dopaminergic denervation, pulsatile L-DOPA administration, downstream changes in striatal proteins and genes, and abnormalities in non-dopamine transmitter systems are factors that contribute to the development of these unwanted side effects6,8,9,10.
The neurotoxin 6-hydroxydopamine (6-OHDA) is a well-characterized tool to study PD in rodents11,12,13,14. Since 6-OHDA does not cross the blood-brain barrier, it must be injected directly into the nigrostriatal pathway. 6-OHDA-induced dopaminergic depletion is concentration- and site-dependent15. Unilateral administration of 6-OHDA at the medial forebrain bundle (MFB) can produce severe (&#62;90%) nigrostriatal damage in rodents16,17,18,19. Chronic administration of L-DOPA to severe unilaterally 6-OHDA-lesioned rodents causes the appearance of dyskinetic-like movements named abnormal involuntary movements (AIMs). Dyskinetic-like movements in rodents share similar molecular, functional, and pharmacological mechanisms related to LIDs in PD patients5. Therefore, 6-OHDA-lesioned rats20 and mice21 are valuable preclinical models to study LIDs. When treated chronically (7-21 days) with therapeutic doses of L-DOPA (5-20 mg/kg), unilaterally 6-OHDA-lesioned rats and mice show a gradual development of AIMs that affect the forelimb, trunk, and orofacial muscles contralateral to the lesion17,18,19,20,22,23,24. These movements are presented at a time course similar to L-DOPA-induced peak-dose dyskinesias in PD patients25 and are characterized by hyperkinetic movements and dystonia5. AIMs are usually scored based on their severity (e.g., when a specific AIM is present) and amplitude (e.g., characterized by the amplitude of each movement)5,23,25.
6-OHDA-lesioned rodent models of LIDs present face validity (i.e., the model has several characteristics that look like the human condition)5,11,26,27,28. Rodent AIMs, similar to what occurs in PD patients, are seen as hyperkinetic (forelimb and orolingual) and dystonic (axial) movements29 and mimics peak-dose dyskinesia. At the molecular and functional level, rodent models share many pathological characteristics with PD patients5, such as upregulation of FosB/&#916;FosB19,26,30,31,32,33 and serotonin transporter (SERT)34,35. Concerning predictive validity, drugs that reduce LIDs in PD patients (e.g., the N-methyl-D-aspartate (NMDA) receptor antagonist amantadine) present antidyskinetic efficacy in the rodent model22,36,37,38,39.
The rodent AIMs rating scale was created based on four AIMs subtypes that include AIMs affecting the head, neck, and trunk (axial AIMs), hyperkinetic forelimb movements (limb AIMs), and dyskinetic-like orolingual movements (orolingual AIMs). Although contralateral rotation (locomotive AIMs) is also present in unilaterally lesioned rodents20,22,23,25,40, it has not been scored as a dyskinetic-like movement since it may not represent a specific measure of LIDs22,37,41.
Here, we will describe how to induce and analyze dyskinetic-like movements (axial, limb, and orolingual AIMs) in the severe (&#62;90%) unilaterally 6-OHDA-lesioned rat model of PD. We organized our protocol based on the previous literature and our laboratory expertise.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-hydroxydopamine hydrobromide</td>
			<td>Sigma-Aldrich, USA</td>
			<td>H6507</td>
			<td>Neurotoxin that produces degeneration of catecholaminergic terminals</td>
		</tr>
		<tr>
			<td>Benzerazide hydrochloride</td>
			<td>Sigma</td>
			<td>B7283</td>
			<td>Peripheral dopa-decarboxylase inhibitor</td>
		</tr>
		<tr>
			<td>Camera Bullet IR Turbo HD (HD-TVI)&#160; 2.8mm B</td>
			<td>HIKVISION</td>
			<td>DS-2CE16C0T-IRP</td>
			<td>Camera used to record all behavior</td>
		</tr>
		<tr>
			<td>Imipramine hidrochloride</td>
			<td>Alfa Aesar</td>
			<td>J63723</td>
			<td>Norepinephrine transporter inhibitor (NET) used to protect noradrenergic neurons from 6-OHDA</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Ceva Animal Health</td>
			<td></td>
			<td>Anesthesia for surgical intervention</td>
		</tr>
		<tr>
			<td>L-3,4-dihydroxyphenylalanine (L-DOPA) methyl ester (hydrochloride)</td>
			<td>Cayman Chemical Company</td>
			<td>16149</td>
			<td>Dopamine precursor</td>
		</tr>
		<tr>
			<td>Mirrors</td>
			<td></td>
			<td></td>
			<td>Used to observe the behavior of animals during experiments in all directions</td>
		</tr>
		<tr>
			<td>Needles 0.30 x 13 mm</td>
			<td>PrecisionGlide</td>
			<td></td>
			<td>Needles used to inject drugs</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Samtec</td>
			<td></td>
			<td>Salt</td>
		</tr>
		<tr>
			<td>Syringes 1 ml Sterile</td>
			<td>BD Plastipak</td>
			<td></td>
			<td>Syringes used to inject drugs</td>
		</tr>
		<tr>
			<td>Transparent cylinders</td>
			<td></td>
			<td></td>
			<td>Used to record animal behavior during experiments</td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride</td>
			<td>Ceva Animal Health</td>
			<td></td>
			<td>Sedative, analgesic and muscle relaxant for surgical intervention</td>
		</tr>
	</tbody>
</table>

rating l-dopa-induced dyskinesias in the unilaterally 6-ohda-lesioned rat model of parkinson's disease

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). The most common pattern observed in the clinic is the peak-dose dyskinesia which consists of clinical manifestations of choreiform, dystonic, and ballistic movements. The 6-hydroxydopamine (6-OHDA) rat model of PD mimics several characteristics of LIDs. After repeated L-DOPA administration, 6-OHDA-lesioned rats exhibit dyskinetic-like movements (e.g., abnormal involuntary movements, AIMs). This protocol demonstrates how to induce and analyze AIMs in 6-OHDA-lesioned rats with 90%-95% dopaminergic depletion in the nigrostriatal pathway. Repeated administration (3 weeks) of L-DOPA (5 mg/kg, combined with 12.5 mg/kg of benserazide) can induce the development of AIMs. The time course analysis reveals a significant increase in AIMs at 30-90 min (peak-dose dyskinesia). Rodent models of LIDs are an important preclinical tool to identify effective antidyskinetic interventions.

Although the AIMs patterns observed in rats are simpler and limited compared to those observed in humans and nonhuman primates, this model reproduces both hyperkinetic and dystonic-like movements induced by chronic L-DOPA administration. Here we present data collected from a group (n = 10) of unilaterally 6-OHDA-lesioned rats chronically treated with L-DOPA (5 mg/kg combined with 12.5 mg/kg of benserazide) for 3 weeks (Monday to Friday). Note that the data presented in Figure 2, <strong clas...

Watch this Scientific Journal Video about Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease at JoVE.com

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease

Rodent models of L-DOPA-induced dyskinesias are invaluable tools to identify therapeutic interventions to attenuate the development or alleviate the manifestations that emerge due to the repeated administration of L-DOPA. This protocol demonstrates how to induce and analyze dyskinetic-like movements in the unilaterally 6-OHDA-lesioned rat model of Parkinson's disease.

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). ...

Rating L-DOPA-induced dyskinesias in the 6-hydroxydopamine rat model of the Parkinson's disease is an important preclinical tool to identify effective antidyskinetic interventions. This model is relatively simple, presents low cost, and has similarities to what happens in the clinic. The procedure will be demonstrated by Danilo Leandro Ribeiro, a PhD student in the laboratory.Begin the experiment with Sprague-Dawley male rats, weighing 200 to 250 grams. House two to three animals per cage under the standard laboratory conditions with food and water available ad libitum. Before the surgery, administer the norepinephrine transporter inhibitor, imipramine, to the rat intraperitoneally.After confirming the anesthesia by the lack of response to toe pinch, position the rat in a prone position in the stereotaxic apparatus on top of the heating pad. Once the rat is positioned, use a scalpel to make a one-centimeter-long incision at the region where the microinjection will occur. Then, clean the skull region with cotton swabs and ensure the bregma and lambda are exposed.Take the medial forebrain bundle, or MFB, stereotaxic coordinates from bregma at 4.3 millimeters anterior, 1.6 millimeters lateral to the right side, and 8.3 millimeters ventral from the durometer. When the coordinates are decided, administer the 6-hydroxydopamine, or 6-OHDA, unilaterally at a rate of 0.4 microliters per minute in the right MFB, with a 50-microliter Hamilton glass syringe. At the end of the surgery, suture the scalp incision and rehydrate the animal with warm, sterile, 0.9%saline solution subcutaneously, at the dose of 10 milliliters per kilogram.Remove the animal from the stereotaxic frame and place it in a warm recovery cage, while monitoring until consciousness is regained. At four weeks post-lesion, measure the effectiveness of the dopaminergic lesion using a stepping test, by assessing the akinesia of the forelimb contralateral to the lesion. Rats presenting three or fewer adjusting steps with the contralateral forelimb should be included in the study as putative, severely 6-OHDA-lesioned rats.Four weeks after the 6-OHDA lesions, start the chronic treatment on Monday and record abnormal involuntary movements on Wednesday. To do so, place the rat inside a transparent cylinder with 20 centimeters diameter and 40 centimeters height, and allow it to acclimatize for at least 15 minutes. Ensure that the floor is covered with the bedding material and mirrors are positioned behind the cylinder to observe the animal from all possible angles.Position a high-resolution video camera to allow the viewing of axial, limb, and orolingual abnormal involuntary movements. The mirrors behind the cylinder will allow tracking abnormal involuntary movements at a 360-degree angle. The camera can be positioned slightly below the plane at a 15-degree angle from animals to observe orolingual abnormal involuntary movements.When the position is decided, fix the camera directly to the bench. Once the animal is acclimatized for 15 minutes, remove the animal from the cylinder and administer five milligrams per kilogram of freshly prepared L-dopamine or L-DOPA combined with 12.5 milligrams per kilogram of benserazide, subcutaneously. Put the animal back in the cylinder and start a timer to track abnormal involuntary movements.Use a video camera to record the abnormal involuntary movements for 180 minutes post-injection, at 30-minute intervals, to carry out offline scoring. After the six observation periods, the scores must be given over one to two minute epochs and classified as axial, limb, or orolingual. Ensure not to include normal behaviors.The treatment with L-DOPA and benserazide should be continued for three weeks, once daily, from Monday to Friday. In the representative time course analysis, the scores applied to axial, limb, and orolingual abnormal involuntary movements over three weeks of chronic L-DOPA administration, are shown. It was noted that L-DOPA-induced, peak-dose dyskinesias occur between 30 to 90 minutes, with a gradual decrease after 120 minutes post-injection.Additionally, the sum of axial, limb, and orolingual abnormal involuntary movements was presented on individuals, scoring days and weekly over three weeks of chronic L-DOPA administration. The abnormal involuntary movements protocol is usually for the investigation of new therapeutic tools with antidyskinetic translational potential.

rating-l-dopa-induced-dyskinesias-unilaterally-6-ohda-lesioned-rat

Research

JoVE Journal

Biology

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...