Analysis of NK1r and NMDAr expression in dorsal root ganglia neurons in the presence of nerve growth factor (NGF) and prostaglandin E2 (PGE2)

ANALYSIS OF NK1r AND NMDAr EXPRESSION IN DORSAL ROOT GANGLIA NEURONS IN THE PRESENCE OF NERVE GROWTH FACTOR (NGF) AND

PROSTAGLANDIN E2 (PGE2)

JAIRO BAQUERO BUITRAGO BIOLOGY SCIENCE M.Sc. CANDIDATE

PONTIFICIA UNIVERSIDAD JAVERIANA FACULTAD DE CIENCIAS BÁSICAS

PROGRAMA DE POSTGRADO BOGOTÁ

ANALYSIS OF NK1r AND NMDAr EXPRESSION IN DORSAL ROOT GANGLIA NEURONS IN THE PRESENCE OF NERVE GROWTH FACTOR (NGF) AND

PROSTAGLANDIN E2 (PGE2)

JAIRO BAQUERO BUITRAGO

Trabajo de Grado presentado para optar el título de: Magíster en Ciencias Biológicas

DIRIGIDO POR:

ESPERANZA RECIO PINTO PhD.

Associate Professor

New York University School of Medicine Departments of Anesthesiology and Pharmacology

PONTIFICIA UNIVERSIDAD JAVERIANA FACULTAD DE CIENCIAS BÁSICAS

PROGRAMA DE POSTGRADO BOGOTÁ

NOTA DE ADVERTENCIA

ANAL YSIS OF NKlr AND NMDAr EXPRESSION IN DORSAL ROOT GANGLIA NEURONS IN PRESENCE OF NERVE GROWTH FACTOR (NGF) AND

PROST AGLANDIN E2 (PGEz)

JAIRO BAQUERO BUITRAGO

APROBADO POR

DIRECTORA

ESPERANZA RECIO PINTO PhD. Associate Professor

New York University School of Medicine Departments of Anesthesiology and Pharmacology

JURADO Dra.lSMAEL SAMUDlO Departamento de Nutrición y Bioquimica

Facultad de Ciencias

Poot;!;daUo;_,;." Jave';ac~

JURADO

Dr. LUDIS MORALES , Departamento de Nutrición yBioquimica

Facultad de Ciencias Pontificia Universidad Javeriana

Dra. INGRID SCHULER Decana Académica Facultad de Ciencias Pontificia Universidad Javeriana

JURADO Dr. JAIME CASTELLANOS

Instituto de Virologia Universidad El Bosque

Dr. MANUEL FRANCO Director de Posgrado

DEDICATION

I dedicate all this work to God who was the one that always stayed with me. He let me

came to the Anesthesiology laboratory in New York University, Langone Medical Center

to give a grain of sand in the research of wellness to patients whom suffer pain in all its

forms.

To my grandmother Azucena Ramirez de Buitrago who always trust in me, to all my

family that always stayed by my side, giving me support and strength to keep on

working in order to succeed in this project.

To my Ex-wife Luz Mila, who one day gave me the push to start doing the master and

helped during this long way to here.

To all the scientists that in any way this information will be valuable in any of their

ACKOWLEDGMENTS

I want to thank first to GOD who is the owner of all the knowledge and let us give a light

in the pain research. To all the people who were with me during this time of effort and

constancy, from good to hard times.

To Dr JJ Thomas Blanck who gave me the chance to belong to his powerful and strong

research group, from the department of Anesthesiology in NYU LMC.

To Dr Esperanza Recio-Pinto who during this two and a half years dedicate her

patience to teach me and guide me in the field of research.

To Dr Michael Dubois MD, who granted and provide the funds for this research in the

New York University, Langone Medical Center.

To my friends and people that collaborate in de development of this project: Jhon Jairo

Sutachan PhD (candidate), Jose Vicente Montoya MD, Martha Salas PhD (Candidate),

Monica Norchini PhD, Alexandra Sideris PhD, Wai Si Chan PhD, Jin Zhang MD, PhD,

Fang Xu PhD, Sonia Luz Albarracin PhD, Maria Fernanda Gutierrrez, Eduardo Baquero

B. DMD., Ricardo Buitrago MD, Edilma Bernal and all the people who walked with me in

TABLE OF CONTENTS

DEDICATION

ACKOWLEDGMENTS

TABLE OF CONTENTS

ABSTRACT

1. INTRODUCTION

2. STATE OF ART

2.1. DORSAL ROOT GANGLION

2.2. NERVE FIBER STRUCTURE

2.2.1. CLASSIFICATION OF NEURONAL SUBPOPULATIONS

2.3. PERCEPTION OF SOMATIC STIMULATION

2.4. SUBSTANCE P

2.5. NEUROTRANSMITERS FROM THE FAMILY OF TACHYKININS 2.6. TACHYKININ RECEPTORS

2.6.1. NK1 RECEPTORS

3. MATERIALS AND METHODS

3.1. DORSAL ROOT GANGLIA (DRG) ISOLATION AND PREPARATION OF DISSOCIATED DRG CELL CULTURE (PRIMARY CELL CULTURE)

3.1.1. DRG ISOLATION

3.1.2. ANIMAL PROCEDURE

3.1.3. DORSAL ROOT GANGLIA NEURON’S CULTURE

3.2. IMMUNOSTAINING

3.3. WESTERN BLOT PROTOCOL

3.3.1. PROTEIN EXTRACTION FROM CELL CULTURE

3.3.2. PROTEIN DETERMINATION

3.3.3. GANGLIA PROTEIN ISOLATION

3.3.4. WESTERN BLOT

3.4. ANALYSIS OF RECEPTOR EXPRESSION.

3.4.1. IMMUNOSTAINING ANALYSIS.

3.4.2. WESTERN BLOT ANALYSIS

3.4.3. STATISTICAL ANALYSIS.

4.1. STANDARDIZATION OF THEDORSAL ROOT GANGLION NEURON CULTURE

4.1.2. DETERMINATION OF THE CONCENTRATION FOR THE TREATMENTS

4.1.3 VARIATION OF THE NK1R EXPRESSION BY TIME

4.1.4. DETERMINATION OF THE EFFECT OF ANIMAL PERFUSION ON DRG NEURON CULTURE IN THE EXPRESSION OF NMDA AND NK1R

4.1.5 DETERMINATION OF THE BACKGROUND.

4.2 ANALYSIS OF THE RECEPTOR EXPRESSION

4.2.1. NEUROKININ 1 RECEPTOR (NK1R)

4.2.2 NMDA RECEPTOR (NMDAR)

4.3. VALIDATION OF THE VARIABILITY IN THE EXPRESSION OF NK1 AND NMDA (NR2B) RECEPTORS

BYWESTERN BLOT

EXPRESSION OF NK1 AND NMDA (NR2B) RECEPTORS BY WESTERN BLOT

4.3.1. NK1 RECEPTOR.

4.3.2. NMDA RECEPTOR. SUBUNIT NR2B

5.1. STANDARDIZATION OF THE NEURON CULTURE

5.1.1. CULTURE MEDIUM

5.1.2. DETERMINATION OF THE TREATMENT CONCENTRATIONS

5.1.3. TIME DETERMINATION

5.1.4. DETERMINATION OF THE PERFUSION METHOD FORNEURON CULTURE

5.2. ANALYSIS OF RECEPTORS

5.2.1 NEUROKININ 1 RECEPTOR (NK1R)

5.2.2 NMDA RECEPTOR

5.3. VALIDATION OF THE VARIABILITY IN THE EXPRESSION OF NK1 AND NMDA (NR2B) RECEPTORS

BYWESTERN BLOT

5.3.1. NK1 RECEPTOR.

INDEX OF FIGURES

Figure 1: Spinal cord, and dorsal root ganglion; sec HE.

Figure 2 Shows the areas of more interest during nociception in pain modulation.[12] Figure 3.Representation of the structure of the Neurokinin receptor 1. Figure 4. NR1 subunit structure representation from the NMDA receptor.

Figure 5: Glutamate receptor classification.

Figure 6. 24 multiwell for cell culture, using 13mm coverslips PDL precoated.

Figure 7.The cassette for protein transfer.

Figure 8. The case, from Biorad Company. Figure 9. Cell sizes from DRG’s.

Figure 10. There is one photomicrography in five different panels. Figure 12. Example of excel data sheet used to collect raw data from the slides.. Figure 13. ImageJ program screen. Membrane image to analyze. Figure 14. Selection of the same area from the bands per treatment.

Figure 15. Area under the peak representation..

Figure 16. Comparison: Neurobasal A (NB) versus DMEM media. Figure 23. NK1r expression in double staining for one experiment using anti-NK1r antibody. Figure 24. General intensity for NK1r in six experiments for NK1r. Figure 27. General intensity for NMDAr in the experiments for NR1 subunit.

Figure 28. NR1 receptor expression by cell size.

Figure 33. NR2B subunit, comparison by cell size.

Table 1. Afferent fiber groups classification. Nerve type IV belongs to the unmyelinated fibers. Table 2. Mammalian and non-mammalian Tachykinins [19]

Table 3. Values for pure Optiprep.

Table 4. Volume of dH2O vs BSA

ABSTRACT

The Anesthesiology Laboratory from The New York University, Langone Medical Center investigates the role of Substance P (SP) in regulating the NMDA-evoked increase in cytoplasmic Ca2+ ([Ca2+]cyt) present in primary sensory Dorsal Root Ganglia (DRG)

neurons, as a possible mechanism by which DRG neurons may become hyper-excitable following injury. Preliminary studies showed that in tissue culture, DRG neurons require both nerve growth factor (NGF) and prostaglandin E2 (PGE2) in order to

get a NMDA-evoked increase in [Ca2+]cyt . Hence, my project consisted in investigating

whether the ability of these neurons become NMDA responsive was due to an increase in NMDA receptors and/or due to neurokinin receptors.

NGF and PGE2 have been previously shown to increase during injury and to contribute

in neuronal sensitization. In order to evaluate changes in the expression of NMDA and Neurokinin receptors in cultured adult rat DRG neuron, we used two different methods: Immunostaining and Western Blotting.

For the immunostaining: we prepared single cell cultures of DRG neurons exposed to three different treatments: the control, which is the culture media; the addition of PGE2

and NGF independently and simultaneously. We used specific primary antibodies against the Substance P (SP) receptor, the NK1r, and for the subunits NR1 and NR2B in N-methyl D-aspartate (NMDA) receptor. We found that treatment with both factors NGF+PGE2, showed a slight increase in the expression of the NK1r as compared to the

control group. About NMDAr’s subunits, the NR1 does not show any significant increase in its expression under any of the treatments. The NR2B subunit shows an increase in expression in the medium size DRG neurons in all the treated groups compared to control.

1. INTRODUCTION

The sensation of Pain is elicited by many different molecular mechanisms. In a group of factors that mediate in pain, one of these mechanisms involved the increase production of substance P (SP). SP released from DRG central nerve terminals enhances the excitability of spinal dorsal horn neurons. The N-Methyl- D-Aspartate (NMDA) that is an intrincated protein involved in the activation of the NMDA receptor, could be studied by applying NMDA, in primary afferent neurons in rats. Several studies indicate that substance P (SP) may act within the DRG to modulate the transmission of nociceptive information by increasing the excitability of DRG neurons, which can also be time-dependent and regulated by the Neurokinin 1 receptor (NK1r), the SP receptor. Studies have reported that there may exist an interaction between NMDAr and NK1r in presence of substance P in the primary sensory neurons and also induced a long-term potentiation on glutamate-induced currents in the dorsal horn neurons [1]. These findings might account for the symptoms of chronic pain including hyperalgesia, Allodynia and spontaneous pain [2].

The aim of the present study is to evaluate whether prostaglandin E2 (PGE2) and Nerve

Growth Factor (NGF) as inflammatory factors, improve the DRG neuronal

responsiveness to NMDA in part by enhancing the expression of the N-methyl D-Aspartate (NMDA) and the Neurokinin 1 (NK1) receptors in primary neuron culture from Dorsal Root Ganglia in adult rats. To end this aim, this work is divided into three parts:

i.The standardization of primary cell cultures. ii. The determination of the optimal concentration for NGF and PGE2, and the best time of exposure to these factors. iii.The

determination of NMDAr and NK1r expression in presence of NGF and PGE2 using

2. STATE OF ART

The human body has the ability to live in every kind of environments. From, the most

appropriate to the extreme ones. This remarkable adaptation is only possible due to a

chain of processes that can occur in the intricate organization in the nerve system as a

whole. The input for the adaptation starts in the sensory system that is in charge to

receive, process and relay stimulus from the exterior to the internal systems in the body.

One of these stimuli could be the pain and all the situations related with the production

of this sensation which are received through an intricate organization that can be

represented by two parallel pathways: one pathway for tactile sensation that is called

the dorsal column-medial lemniscal system; and the other that is the anterolateral

system which main function is to mediate pain. The process starts in the exterior of the

body and is first register for the sensitive neurons present in the skin, which is the

border of the body, and connected to the first neurons in the chain of events that goes

to the central nervous system (CNS). Those primary afferent neurons are the Dorsal

Root Ganglia (DRG) neurons. In which anatomically, only the distal portion of the

peripheral axonal branch is specialized to encode stimulus energy. The remaining

portion of the axon is specialized in conducting information, encoded in form of action

potentials, and to transmit that to the central nervous system.

Sensory systems are serially and hierarchically organized like a very intricate

organization. Receptor neurons, the first neuron order for the somatic sensory system

converges onto second-order neurons in CNS. Second and third order of neurons does

have a specific function carrying the information to the spinal cord and then to the

Thalamus and the way back to specific response. Other complicated functions like

movement are further controlled and modulated by afferent projections to supraspinal

structures such as the cerebellum.[4]

2.1. Dorsal Root Ganglion

The Dorsal Root Ganglion (DRG) neurons are a group of primary sensory neurons in a

inserted in the intervertebral foramina, just proximal to the union of the dorsal and

ventral roots. [5] They are named based on the region were they are located along the

vertebral column. DRGs contain cell bodies of neurons from afferent spinal nerves.

Dorsal root ganglia and ganglia of cranial nerves are involved in general sensation, and

have the same histological structure. Primary afferents neurons in vertebrates are

pseudounipolar, and the central projections from the afferent neurons terminate in the

dorsal horn of the spinal cord and the peripheral projections end in the skin, muscles,

tendons, joints and internal organs. Peripheral processes of primary afferent neurons

are among the longest axons in the body (about one meter in humans) and are usually

classified into myelinated (Aδ) and unmyelinated axons with small diameter called fibers

type C. Although both fiber types Aδ and C can transmit nociceptive messages from the

periphery to the spinal cord, C-fibers are usually considered the dominant “pain” fibers.

In a piece of ganglia the most common population of cells are the neurons, then

fibroblasts and then satellite and Schwann cells as shown in Figure 1.

2.2. Nerve fiber structure

The basic structure of a nerve fiber is an axon, the myelin sheath (for fibers type A and

B), and the neurolemma (sheath of Schwann); its plasma membrane is named

axolemma, but the neurolemma and the myelin sheath made part of Schwann cells. The

myelin sheath is interrupted at intervals between 100µm to about 1mm, depending on

the length and thickness of the fiber. Those intervals are named Nodes of Ranvier, and

there is one Schwann cell by internodes. The internodal parts of the axon are insulated

by the myelin sheath, but they do not cover the entire contact surface, leaving an empty

space between the cells through which the axolemma at the node is in contact with

extracellular fluids, and voltage-gated sodium channels that are present only at nodes.

This arrangement allows action potentials to skip electrically from node to node along

myelinated fibers calling this action salutatory conduction.[5]

DRGs’ are related with the Somatosensory systems, which comprising the receptors

and processing centers to produce the sensory modalities such as touch, temperature,

Proprioception (body position), and nociception (pain). The pathway from external

stimuli follows the cell bodies of all multipolar neurons axons, the afferent neurons out of

the CNS. They bring the somatosensory information to the spinal cord through DRG

neurons. All those nerves which join a dorsal root make a spinal nerve. Those axons

that leave the spinal cord trough the ventral roots control muscles and glands and they

are referred to as efferent axons because they “bear away from” the CNS. This

processing primarily occurs in the first Somatosensory area in the parietal lobe of the

cerebral cortex.[7]

2.2.1. CLASSIFICATION OF NEURONAL SUBPOPULATIONS

All the neurons from DRG have morphological, biochemical and functional differences,

which let them be classified in populations and eventually subpopulations; probably

these differences could be genetic or epigenetic. [8]

The first neuronal classification in rat was based on the neuronal body size in the light

microscope, dividing them into two main types: neurons type A (big and light), and type

B (small and dark). They are also classified in subpopulations based on the Nissl

Also neurons type A presented clusters in the RER in the peripheral pericardia,

separated by thin threads of neuroplasm, otherwise in the type B neurons, the RER

remains compact and free of cytoplasmic spaces. At the same time every neuronal type

was divided into subtypes (A1, A2, A3 and B1, B2, B3) based on the distribution of the

NS in the cytoplasm. From the Golgi apparatus structure and its staining patterns with

Iodine Osmium and zinc, another classification was done, adding a third group, the

neurons type C, the smallest inside the ganglion. [9]

The primary afferent nerve fibers are classified based on where it comes from; they can

be classified in muscular, cutaneous and visceral. In all of them myelinated fibers type I,

II and III can be found and also unmyelinated fibers type IV. There are a very close

relationship between the stimuli and the kind of fiber that conducts the stimuli. The pain

and the temperature (nociception and thermal sensitization) are conducted by fibers

type III and IV, the mechanical ones (mechanoreceptors) by types IV and the

proprioceptive by types I and II. [10]

The diameter of the fiber determines the speed at an afferent fiber conducts the action

potential. In large myelinated fibers, the conduction velocity (CV), in meters per second,

is approximately equal to six times the diameter (in micrometers). The statistical

representation of studies showed that responses correspond to four peaks: large

myelinated (I), small myelinated (II), smaller myelinated (III) and unmyelinated (IV)

fibers. Rather than adhere to this numerical classification, which is used for muscle

afferents, physiologist that studied cutaneous nerves chose another nomenclature: Aα,

Aβ, Aδ, and C. Cutaneous nerves has not Aα afferent nerves. The CV of a fiber has

important functional significance. The faster a fiber conducts action potentials, the

quicker the CNS receives the information. Consider that in an average adult a stimulus

delivered to a finger tip activates receptors that are located about 1 m from the spinal

cord. An Aβ fiber, conducting at 50 m/sec, conveys its information to the CNS in 0.02

sec. In contrast, a C fiber, conducting at the rate of 0.5 m/sec, takes 2 sec or more to

convey the information it carries to the CNS (see table 1). If the stimulus is noxious and

receives the information. Time delays also occur in the central processing of a stimulus,

which further increase the possibility of damage.

Muscle nerve

Cutaneous nerve

Fiber diameter (μm)

Conduction Velocity (m/sec)

I 13-20 80-120

II Aβ 6-12 35-75

III Aδ 1-5 5-30

IV C 0.2-1.5 0.5-2

Table 1. Afferent fiber groups classification. Nerve type IV belongs to the unmyelinated fibers.

2.3. Perception of somatic stimulation

The perception of most of the stimuli are perceived by basically three major classes of

somatic receptors, the mechanoreceptors, thermoreceptors, and nociceptors.

Any changes in pressure in the body skin can produce one of two responses based on

the speed of the conduction of the impulse through the nerve: the slow adapting

mechanoreceptor that responds continuously to an enduring stimulus and the faster that

only answer to the onset of long-lasting stimulus can produce. There is a specialized

group of neurons that form corpuscles that work also as mechanical receptors.

Glabrous skin contains two kinds of rapidly adapting mechanoreceptors: The Meissner

corpuscle located in the dermal papillae, and the Pacinian corpuscle located in the

subcutaneous tissue to register stimuli like pressure. There are two more corpuscles:

Nerkel and Ruffini for specific perception but speeds up the transmission of small

sensations.[4]

There are moderate or constant stimulus that applied to the skin fails to produce any

sensation after it has been presented for a while. The threshold of each stimulus can

adaptation. This adaptation occurs because of the physical construction of the skin and

the cutaneous sensory organs. [7] Confirming the law of specific energies first

formulated by Muller, it is known that different mechanoreceptors mediate different

sensations. Natural stimuli rarely activate a single type of receptor; they activate

different combinations of mechanoreceptors. Proprioception is the sense of balance,

position, and movement. Balance is largely mediated by the specialized receptors of the

vestibular apparatus.

There are other modalities of somatic sensations that are controlled by the peripheral

receptors. As in the mechanoreceptors, feeling of warmth and coolness are relative, not

absolute except for the extremes. There is a temperature level that, for a particular

region of skin, will produce a sensation of temperature neutrality, neither warmth nor

coolness. This level is named threshold and depends on the history of the region and

how often the stimuli was applied to it. Cold receptors are connected to fibers that

belong to the same bands of the fiber spectrum as the pain fibers, the Aδ and C fibers.

The cold fibers discharge intensely when a cold stimulus is delivered, and the frequency

of firing is proportional to the rate and extent of temperature lowering. Thermal

receptors, like nociceptors, are free nerve ends. Nerve fibers are totally related to the

body temperature kinetics between warmth and heat pain. Warmth is mediated by

unmyelinated C warm fibers, whereas the initial pain associated with intense heat is

mediated by heat nociceptors (Aδ). Therefore, for warm stimuli (less than 45oC), there is a strong correlation between the discharge properties of warm fibers and estimations of

warmth, but the correlation is only weak for hot stimuli (greater than 45oC). [4]

The receptors that correspond selectively to damaging stimuli are called nociceptors

(Latin nocere, to injure). Nociceptors are a subpopulation of sensory neurons that are

activated by “noxious” stimuli that can produce tissue damage. Compelling evidence

suggests that plasticity in nociceptors contributes substantially to the increase pain one

feels in the presence of injury. Plasticity in nociceptors is critical for both the

development and maintenance of plasticity in the CNS[11]. Nociceptors are connected

to axons that belongs to two fiber classes: Aδ and C. There are three main types of

stimulation and most effectively by sharp objects. No response is evoked in this type of

nociceptors when a blunt probe is pressed firmly into the skin, but a pinprick or pinch

causes a brisk response. 2. Heat nociceptors respond when the receptive field is heated

to temperature greater than 45oC, the heat pain threshold in humans. 3. Polymodal

nociceptors that respond equally to all kinds of noxious stimuli-mechanical, heat, and

chemical. Morphologically, nociceptors are bare nerve endings, it is not known,

however, whether nociceptors respond directly to the noxious stimulus or indirectly by

means of one or more chemical intermediaries released from the traumatized tissue.

It is good to understand that our awareness of pain and our emotional reaction to it is

controlled by mechanisms within the brain. Our brain possesses mechanisms that can

reduce pain, partly through the action of the endogenous opioids. Another type of

nociceptive fiber contains receptors that are sensitive to ATP (Burnstock and Wood,

1996). These receptors are ionotropic and control channels that admit sodium and

calcium ions. ATP is also released when the blood supply to a region of the body is

disrupted (a condition called ischemia, which occurs during spasms of blood vessels

that causes angina or migraine) or when a muscle is damaged. It is also related by

rapidly growing tumors. Thus, these nociceptors may be at least partly responsible for

the pain caused by angina, migraine, damage to muscles or cancer.[7]

There are also many factors that can produce pain. One example can be the related

with the VR1 receptor, a pain receptor that is sensitive to capsaicin, heat, and acids,

which appears to be involved in the pain caused by inflammation, that often

accompanies injuries to skin or muscle, greatly increases sensitivity of the inflamed

region to painful stimuli, this effect motivates the individual to minimize movement or the

injured part and avoid contact with other objects.

There are two different types of pain: fast and slow. Fast pain is an abrupt and sharp

sensation that is carried by Aδ fibers that have lightly myelinated axons, conducting

action potentials rapidly, and have medium to large diameter cell bodies. A-fibers

mediate the fast, pricking quality of pain. [11] Slow pain, carried by C fibers, is a

sickening burning sensation, which follows fast pain. C-fibers have unmyelinated axons,

the slower, burning quality of pain. C-fibers comprise around 70% of all nociceptors.

Two classes of C-fibers have been identified; one class contains a variety of

neuropeptides, including substance P and calcitonin gene-related peptide, and

expresses TrkA receptors, the high affinity receptor for nerve growth factor. These

neurons project o the outermost region of the spinal dorsal horn (lamina I and outer

lamina II) and terminate largely on spinal neurons that project to higher order pain

centers in the brain. The other class contains few neuropeptides but expresses a

surface carbohydrate group that selectively binds to a plant lectin called isolectin B4

(IB4). This subpopulation of neurons (spinal neurons) is supported by glial-derived

neruotrophic factor during early postnatal development. The IB4 binding neurons project

to a different region of the spinal dorsal horn (inner lamina II) that contains primarily

local spinal interneurons. [11] Damaged tissues can sensitize the C-fibers causing the

release of algesic mediators such as prostaglandins, potassium, histamine,

leukotrienes, bradikinin, and substance P. This is the rationale for the use of systemic

no steroidal anti-inflammatory drugs and aspirin, which decrease the production of

sensitizing prostaglandins in patients who have acute inflammatory pain.[12] [13]

Pain seems to have three different perceptual and behavioral effects. First is the

sensory component, the pure perception of the intensity of a painful stimulus. The

second component is the immediate emotional consequences of pain, the

unpleasantness or degree to which the individual is bothered by the painful stimulus.

And the third component is the long-term emotional implications of chronic pain, the

threat that such pain represents to one’s future comfort and well-being. These three

components of pain appear to involve different brain mechanisms. The purely sensory

component of pain is mediated by a pathway from the spinal cord to the ventral

posterolateral thalamus to the primary and secondary somatosensory cortex. The

immediate emotional component of pain appears to be mediated by pathways that

Figure 2 Shows the areas of more interest during nociception in pain modulation.[12]

A particularly interesting form of pain sensation occurs after a limb is gone, up to 70

percent of amputees report that they feel as though the missing limb still exists and that

it often hurts. This phenomenon is referred to as the Phantom Limb. People with

phantom limbs report that the limb feels very real, and they often say that if they try to

reach out with it, it feels as though there were responding. Sometimes, they perceive it

as sticking out, and they may feel compelled to avoid knocking it against the side of a

doorframe or sleeping in a position that would make it come between them and the

mattress. People gave reported all sorts of sensations in phantom limbs, including pain,

pressure, warmth, cold, wetness, itching, sweatiness, and prickliness. The classic

explanation for phantom limbs has been activity of the sensory axons belonging to the

amputated limb.[7]

The pain receptors can be stimulated by three different stimuli: mechanical, thermal and

chemical. The response also received the name of mechanical pain, thermal pain and

bradikinin, serotonin, histamine, potassium ions, acids, acetylcholine, and proteolytic

enzymes. In addition, prostaglandins and Substance P enhance the sensitivity of pain

endings but do not directly excite them. The chemical substances are especially

important in stimulating the slow, suffering type of pain that occurs after tissue injury. In

contrast to most other sensory receptors of the body, the pain receptors adapt very little

and sometimes not at all. In fact, under some conditions, the excitation of pain fibers

becomes progressively greater, especially so for sole aching nauseous pain, as the pain

stimulus continues. This inverse in sensitivity of the pain receptor is called hyperalgesia.

One can readily understand the importance of this failure of pain receptors to adapt,

because it allows the pain to keep the person apprised of a tissue-damaging stimulus as

long as it persists.[16]

In theory all pain receptors are free nerve endings, endings that use two different

pathways to transmit pain signals to the CNS, corresponding to two different kinds of

pain: first, the fast sharp pain pathway in the peripheral system, produced by either

mechanical or thermal pain stimuli. It is transmitted by the fibers type Aδ at velocities

between 6 and 30 m/sec. The second is the slow chronic kind of pain that is produced

by chemical types of pain stimuli; this slow chronic pain is transmitted by type C fibers at

velocities between 0.5 and 2 m/sec. The slow pain tends to become greater over time.

This sensation eventually gives one the intolerable suffering of long continued pain and

makes the person continue to try to relieve the cause of the pain. On entering the spinal

cord from the dorsal spinal roots, the pain fibers terminate on neurons in the dorsal

horn.

The substance P is one slow chronic neurotransmitter and is also produced by type C

nerve endings. Research experiments suggest that the type C pain fiber terminals

entering the spinal cord secrete glutamate transmitter and substance P transmitter. The

glutamate transmitter acts instantaneously and lasts for only a few milliseconds.

Substance P is released much more slowly, building up in concentration over a period

of seconds or even minutes, in fact, it has been suggested that the “double” pain

sensation one feels after a pinprick might result partly from the fact that the glutamate

more lagging sensation. Regardless of the yet unknown detail, it does seem clear that

glutamate is the neurotransmitter most involved in transmitting fast pain into the central

nervous system, whereas substance P (and other related peptides) is concerned with

slow chronic pain.[16]

Cuts, scrapes and bruises are kind of painful stimuli related with tissue damage.

Hyperalgesia is the enhanced sensitivity and responsively to stimulation of the area

around the damaged tissue. Thus, in the region surrounding an injury, stimuli that would

not normally cause pain are perceived as painful, and stimuli that would ordinarily be

painful are significantly more so (therefore, hyperalgesia). The cause of this

phenomenon is the sensitization of nociceptors by various substances released when

tissue is damaged. It is evident; that the release of bradykinin, histamine,

prostaglandins, and other agents from the site of injury enhances the responsiveness of

nociceptive endings. Based on the theory there is an electrical activity in the nociceptors

themselves that stimulates the local release of chemical substances like substance P,

that cause vasodilatation, swelling, and the release of histamine from mast cells. Injury

and pain are thus intertwined in a complex cascade of local signals. The involvement of

these substances in the production of pain has also provided clues about how some

analgesics may work, suggesting strategies for pain relief. Aspirin (salicylic acid), for

example, evidently acts by inhibiting cyclooxygenase, an enzyme important in the

biosynthesis of prostaglandins. The presumed purpose of the complex chemical

signaling arising from local damage is not only to protect the injured area as a result of

the painful perceptions produced by ordinary stimuli close to the site of damage, but

also to promote healing and guard against infection by means of local effects such as

increased blood flow and inflammation. [17]

Allodynia that means “other pain” is a painful response to a usual non painful stimulus

and can be either static or mechanical. [19] Allodynia is different from hyperalgesia, an

extreme reaction to a stimulus that is normally painful. There are two different kinds of

allodynia: Mechanical Allodynia that is also known as tactile Allodynia, and present two

pressure and (2) Dynamic mechanical Allodynia, that is pain in response to brushing;

and thermal Allodynia that is pain from mild skin temperatures in one affected area.

2.4. Substance P

Substance P (SP) is a neuropeptide, an undecapeptide that modulates the excitability of

the dorsal horn ganglion. At the cellular level the synthesis of SP occurs in the ribosome

and is confined around the nucleus, then is packed into storage vesicles and axonal

transported to terminal endings for final enzymatic processing. [18] SP is secreted from

the terminals of specific sensory nerves. Biochemical and immunohistochemical studies

demonstrate that Substance P is transported to both the central and the peripheral

branches of primary sensory neurons. However, four times as much

SP-immunoreactivity is accumulated in the peripheral branches of primary sensory neurons.

The largeness of SP is produced in the sensory ganglion and exported towards the

terminal regions of the peripheral branches, at an average speed of 5-6 mm/h by a

mechanism of axonal transport. A number of enzymes are involved in the metabolism of

SP, including: neutral endopeptidase (NEP: metallopeptidase EC); SP-degrading

enzyme (SP-DE); angiotensin-converting enzyme (ACE); dipeptidyl aminopeptide IV

(DP IV); postproline endopeptidase (PEP); cathepsin D (EC-D) and cathepsin-E (EC-E).

Snider et al in 1991 reported the first neuropeptide NK1 antagonist (CP96 345), and in

1997 Betancourt et al listed more than 20 non-peptide NK1 antagonists. Since then,

much more compounds have been developed, with special interest in pharmaceutical

companies.[19]

SP plays an important role in nociception at the spinal level. Substance P is synthesized

by a subpopulation of small diameter sensory neurons, and is released in response to

peripheral inflammation and noxious stimulation. SP can also be released in the dorsal

horn by normally inflammation, by the expression of NK1 mRNA, which increases within

the superficial dorsal horn of the spinal cord. The concept of co-transmission can be

applied to sensory neurons. Substance P and NK1, colocalized in small dorsal root

ganglion (DRG) neurons, excite dorsal horn interneurons and synergistically increase

colocalized neuropeptides may interact functionally. Using specific antagonists of the

NK1 and NK2 receptors, the differential functions of SP and NKA have been

demonstrated in the spinal cord. One example of blocking the NK1 is the antagonist

CP-96-345 that effectively blocks spinal cord transmission following afferent neurons. In

contrast, the NK2 antagonist Menarini 10207 blocks spinal hyperexcitability following

conditioning stimulation of muscle, but not cutaneous afferents. This differential effect

has been confirmed using other specific antagonists. Calcitonin gen related peptide

(CGRP) is another peptide colocalized with Substance P in many DRG neurons.

Substance P, but not CGRP, injected onto the lumbar spinal cord in rats evokes a brief

caudally directed biting/scratching response lasting less than five minutes, but when SP

and CGRP are coadministrated, a much more prolonged and intense biting/scratching

behavior was observed. A third neuropeptide colocalized in DRGs is Galanin, which is

dramatically up regulated after peripheral nerve injury. Galanin has a complex effect on

spinal excitability, but has, especially after nerve injury, an inhibitory function and

functions as an endogenous SP and CGRP antagonist. Thus Sp containing DRG

neurons contain at least two “intrinsic modulators” one enhancer (CGRP) and one

attenuator (Galanin).

Glutamine is the main neurotransmitter in the DRG neuron, which is also colocalized

with SP. Glutamate, plays an important role in wind-up and spinal sensitization. Some

experiments had shown that the prolonged facilitation of the flexor reflex following

activation of C afferents fibers, could be reduced by glutamate (NMDA) antagonist.

Moreover when NMDA and NK1 antagonists are co-administered, both wind up and

spinal hyperexcitability are synergistically reduced, suggesting co-release of glutamate

and SP and synergistic interaction inducing central sensitization. [19]

In 1995 Mantyh and colleagues published a remarkable paper in Science using a highly

sensitive and specific antibody to the NK1 receptor. They demonstrated that the

receptor protein, normally confined to the cell membrane of a neuron population in the

dorsal horn, was dramatically internalized after somatosensory stimulation. This

receptor’s endocytosis was reversible, so within 30 min the immunohistochemical image

the cell membrane. Some authors followed the NK1 receptor internalization as a marker

of SP release. The expression of NK1r in the dorsal horn is modified by peripheral

inflammation. Thus a short noxious mechanical stimulus induces internalization in many

more neurons in lamina I after inflammation than in normal rats, and moreover

internalized receptors extend into deeper layers and show an expanded rostrum-caudal

distribution in rats with inflammation as compared to normal rats. Thus inflammation

causes a “reorganization” of dorsal horn circuits.[20]

The biological actions of SP are mediated by Tachikinin receptors, that will be

referenced in next paragraphs, which belong to rhodopsin-like membrane structure,

consisting of seven hydrophobic transmembrane domains, connected by extra and

intracellular loops and coupled G-proteins.

2.5. NEUROTRANSMITERS FROM THE FAMILY OF TACHYKININS

Some polypeptides are hormones related with neuropeptides, and depending on the

size of the molecule, and the source where they are produced, are related with the

neurotransmitters [17]. The biological activity of the peptide neurotransmitters depends

on their amino acid order. Substance P and the opioids peptides are involved in the

perception of pain. Still other peptides, such as melanocyte stimulating hormone,

adrenocorticotropin, and B-endorphin, regulate complex responses to stress. The large

number of neuropeptides transmitters have been loosely grouped into five categories:

brain and gut peptides, opioids peptides, pituitary peptides, hypothalamic releasing

hormones, and catchall category containing all other peptides no easily classified.[17]

The Tachikinin family of neurotransmitters is divided into three groups: the substance P,

the Neurokinin A and the Neurokinin B (Table 20). The expression of this

neurotransmitters is regulated by two gens the preprotachynin I (PPT I) and the PPT II.

The PPT I gene can express four different forms of mRNA through alternative splicing,

two of which (the B and lambda forms) encode synthesis of both SP and Neurokinin A

(NKA), while the two other, the alpha and sigma forms, encode SP only. The beta and

neuropeptide lambda (NP-λ), which is elongated forms of NKA, although their function

has not been fully clarified. It is the PPT II gene that gives rise to Neurokinin B (NKB).

Tachykinin Sequence

Substance P Arg – Pro – Lys – Pro – Gln – Gln –Phe– Phe – Gly –Leu–Met–NH2 Neurokinin A His – Lys – Thr – Asp – Ser –Phe– Val –Gly–Leu–Met–NH2 Neurokinin B Asp – Met – His – Asp – Phe –Phe Val –Gly –Leu–Met–NH2

Eledoisin pGlu – Pro – Ser – Lys – Asp – Ala –Phe– Ile –Gly–Leu–Met–NH2 Kassinin Asp – Val – Pro – Lys – Ser – Asp – Gln –Phe– Val –Gly–Leu–Met–NH2

Table 2. Mammalian and non-mammalian Tachykinins [19]

2.6. Tachykinin Receptors

There are three types of tachykinin receptors, NK1 related with substance P, NK2

related with Neurokinin A, and NK3 that has affinity with Neurokinin B. However,

endogenous Tachykinins are not highly selective for any given receptor, and all can act

on all three receptors under certain conditions such as receptor availability or at high

peptide concentrations. For this reason SP activates not only NK1 receptors but also

NK2 and NK3 receptors in the tissues.

2.6.1. NK1 Receptors

Although SP is the preferred ligand of NK1 receptors (affinity 0.05 – 0.5 nM), all

Tachikinin, share a common COOH- terminal amino acid sequence, which essentially

dictates their biological activity, show some degree of cross-reactivity among tachykinin

receptors. Cloned NK1 receptors display very high degree of sequence homology

between species including man, mouse, rat, guinea pig. Protein sequencing of human

and rat NK1 receptor shows a 92% similarity between the two species. However, using

selective NK1 antagonists there are discrete variations between species. These

variations have been linked to positions 116 and 290 that contain Val and Ile on the

Figure 3.Representation of the structure of the Neurokinin receptor 1.

The NK1r has a relatively long 5’ untranslated region compared to the other tachykinin

receptors which is preceded by a single TATAAA sequence. Analysis of the 5’

untranslated region reveals a cyclic adenosine monophosphate (cAMP) response

element binding protein (CREB)/Calcium response element sequence at 627 pb, which

is adjacent to the TATAAA sequence. Stimulation of the NK1r produces intracellular

inositol 1,4,5-triphosphate (IP3) turnover with subsequent elevation of intracellular

calcium. Thus the CREB/calcium element would allow the genes to respond to elevated

levels of calcium or cAMP with enhanced gene transcription. This sequence could play

an important role in intense or sustained NK1 stimulation, as NK1 responses are rapidly

desensitized and thus a re-sensitization element could exist. Approximately 30% of the

residues of the cytoplasmic carboxyl-terminal tail of the NK1r are Ser and Thr in nature.

These are potential sites for phosphorylation of the G-protein coupled receptors.

Stimulation of NK1r activates phospholipase Cβ (PLCβ), which results in a transient

increase in IP3 and cytoplasmic calcium concentrations, phospholipase A2 induces an

increase in arachidonic acid mobilization and adenylyl cyclase evokes cyclic adenosine

monophosphate accumulation. However, there appears to be no cross talk between

cAMP accumulation and IP3 formation. In the CNS, expression of NK1r is highest in the

caudate-putamen and superior coliculus, with moderate to low levels of NK1r in the

inferior coliculus, olfactory bulb, hypothalamus, hippocampus, substantia nigra and

the spinal cord. Combination of Immunohistochemistry, fluorescence and confocal

microscopy has been used to identify NK1r localization. In these studies SP can induce

internalization in NK1r neurons in laminae I, II, X and a number of neurons in laminae

III-V of the spinal cord. Furthermore, SP induced internalization was abolished by NK1r

antagonist. In the Peripheral Nervous System (PNS), expression of NK1r has been

demonstrated in rat, and mouse DRGs, intrinsic neurons of the gut and in unmyelinated

axons in glabrous skin.[18]

Over the years it has been suggested that NK1r may function as auto-receptors, thus

proposing that SP modulates its own release. Modulatory activity of SP presynaptically,

has been demonstrated in cat spinal cord, in chick sympathetic and ciliary ganglion,

where exogenous SP can evoke both inhibitory and excitatory effects on the ganglia. A

number of NK1r selective antagonists (RP67580 and SR140333) have been

demonstrated to increase SP release from spinal cord evoked by electrical stimulation

of C-fibers, thus supporting the proposal that SP can exert negative feedback on its own

release via activation of inhibitory NK1 autoreceptors and/or by blocking potassium

channels and thus affecting changes in membrane depolarization. SP could also

enhance its own release via production of inositol 1,4,5-triphosphate and consequently

release of calcium ion form internal stores. The autoreceptor role of SP receptors may

have important implications in pathophysiology concerning inflammation, nerve injury or

noxious stimuli. For example, painful neuropathies which are characterized by

persistent Allodynia and hyperalgesia, may themselves generate an ectopic action

potential which is capable of maintaining nociceptive discharge.[18]

L-Glutamate is the major excitatory neurotransmitter in the mammalian CNS, acting

through both ligand gated ion channels (ionotropic receptors) and G-protein coupled

(metabotropic) receptors. Activation of these receptors is responsible for basal

excitatory synaptic transmission and many forms of synaptic plasticity such as long-term

learning and memory. They are thus also potential targets for therapies for CNS

disorders such as epilepsy and Alzheimer’s diseases.

Scientist found glutamate (glutamic acid) and GABA (gamma-amino butyric acid) from

very simple organisms to more complex ones, concluding that these neurotransmitters

are the first to have evolved. Besides producing postsynaptic potentials by activating

postsynaptic receptors, they also have direct excitatory effects (glutamic acid) and

inhibitory effects (GABA) on axons; thus affecting the rate at which action potentials

occur.[21]

Glutamate is the principal excitatory neurotransmitter in the brain and spinal cord. It is

produced in abundance by the cell’s metabolic processes. There is no effective way to

prevent its synthesis without disrupting other activities of the cell. Investigators have

discovered four types of glutamate receptors. Three of these receptors are ionotropic

and are named after the artificial ligands that stimulate them: The AMPA receptor, the

Kainate receptor and the NMDA receptor, they receive this name by the agonist that

activates them: α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate, kainic acid and

N-methyl D-aspartate, respectively. The other glutamate receptor is the Metabotropic

receptor. Actually, there appear to be at least seven Metabotropic glutamate receptors;

they seem to work as presynaptic autoreceptors. The whole group of ionotropic

glutamate receptors is nonselective cation channels, which permit the flow of Na+ and K+ ions, and in some cases small amounts of Ca2+. The postsynaptic currents produced have a reversal potential near to 0 mV; hence AMPA, Kainate, and NMDA receptor

activation always produces excitatory postsynaptic responses. In the same way than

ligand-gated channel receptors, AMPA, Kainate and NMDA receptors are formed by the

association of protein subunits which can combine in different ways to produce many

isoforms.[17]

The AMPA receptor is the most common glutamate receptor. It controls a sodium

channel, because when glutamate attaches to the binding site, it produces excitatory

postsynaptic potentials, the same occurs with the Kainate receptor. It is composed of

four types of subunits: GluR1, GluR2, GluR3, and GluR4, also called GluRA-D. They can

which consist of symmetric “dimer of dimers” of GluR2 and either GluR1, GluR3 or

GluR4. The dimerization starts in the endoplasmic reticulum with the interaction of

N-terminal LIVBP (N-N-terminal leucine/isoleucine/valine-binding protein) domains; GluR

subunits have an extracellular N-terminus and an intracellular C-terminus. AMPA

receptors are both glutamate receptors and cation channels that are integral to plasticity

and synaptic transmission at many postsynaptic membranes. One of the most widely

and thoroughly investigated forms of plasticity in the nervous system is known a

long-term potentiation or LTP. There are two necessary components of LTP: presynaptic

glutamate release, and postsynaptic depolarization. Therefore, LTP can be induced

experimentally in a paired electrophysiological recording when a presynaptic cell is

stimulated to release glutamate on a postsynaptic cell that is depolarized.[22]

There are five types of kainite receptor subunits GluR5, GluR6, GluR7, KA1 and KA2,

which are similar to AMPA and NMDA receptor subunits and can be arranged in

different ways to form a tetramer, a four subunit receptor. Glur5-7 can form homomers

and heteromers; however, KA1 and KA2 can only form functional receptors by combining

with one for the GluR5-7 subunits. Kainate receptors play a role in both pre and

postsynaptic neurons. They have a somewhat more limited distribution in the brain

compared to AMPA and NMDA receptors and their function is not well defined but KA

receptors are related with epilepsy and sensory transduction.

The NMDA receptor has some special and important characteristics. The NMDA

receptor contains at least six different binding sites: four located on the exterior of the

receptor (Polyamine, Glutamate, Glycine [23] and Zinc ion), and two located deep within

the ion channel (Magnesium ion and PCP). When it is open, the ion channel controlled

by the NMDA receptor permits both sodium and calcium ions to enter the cell. The influx

of both of these ions causes a depolarization of the receptor, but the entry of calcium

Figure 4. NR1 subunit structure representation from the NMDA receptor. The C-terminus is intracellular; there are four hydrophobic regions within the central portion of the sequence (I, II, III and TM-IV, and also a ligand binding domain and N-terminal both located extracellular.

Resent investigations provided important results in alteration in the characteristics of the

synapse that provides one of the building blocks of a newly formed memory. The drug

AP5 (2-amino 5-phosphonomentanoate) blocks the glutamate-binding site on the NMDA

receptor and impairs synaptic plasticity and certain forms of learning. [21] For that to

happen, a molecule of glycine must be attached to the glycine binding site, located on

the outside of the receptor. An additional requirement for the opening of the calcium

channel is that a magnesium ion will not be attached to the magnesium binding site,

located deep within the channel. Under normal conditions, when the postsynaptic

membrane is at the resting potential, a Magnesium ion (Mg2+) is attracted to the

magnesium-binding site and blocks the calcium channel. If a molecule of glutamate

attaches to its binding site, the channel widens, but the magnesium ion still blocks it, so

no calcium can enter the postsynaptic neuron. However, if the postsynaptic membrane

is partially depolarized, the magnesium ion is repelled from its binding site. Thus the

depolarized. The NMDA receptor, then, is a voltage and neurotransmitter dependent ion

channel. If a zinc ion (Zn2+) binds with the zinc binding site, the activity of the NMDA receptor is decreased. On the other hand, the polyamine site has a facilitator effect.

Polyamines are chemicals that have been shown to be important for tissue growth and

development. The PCP site, located deep within the ion channel near the magnesium

binding site, binds with a hallucinogenic drug PCP (Phencyclidine, also known as “angel

dust”). PCP serves as an indirect antagonist; when it attaches to its binding site, calcium

ions cannot pass through the ion channel. PCP is a synthetic drug and is not produced

by the brain. Thus, it is not the natural ligand of the PCP binding site.[21]

3. MATERIALS AND METHODS

3.1. DORSAL ROOT GANGLIA ISOLATION AND PREPARATION OF DISSOCIATED DRG CELL CULTURE (Primary cell culture)

3.1.1. DRG isolation

3.1.1.1. Primary cell culture.

During the neuron culture two different kinds of containers were used, one was a tissue

culture Dish (Falcon ref. 35-3001 35 X 10 mm. Becton Dickinson), in which cells were

cultured directly on the dish and used for protein collection to be used in Western Gels.

The other one was a multi 24 well plates (Falcon 35-3047. Multiwell, 24 well tissue

culture Plate, Becton Dickinson) containing round glass coverslips (diameter of 13 mm).

In both cases the culture dishes and the coverslips were pretreated with poly-D-lysine

(PDL) from Sigma (Poly-D-Lysine hydrobromide. SIGMA P6407. CAS 27964-99-4). The

day before the cell isolation 2 ml of PDL (12.5 µg/ml) were put in each tissue culture

dish or 500 µl of PDL were put on top of each coverslip; and incubated for three hours

at room temperature (RT). Then the dishes or coverslips were washed four times with

deionized and sterile water. Then the dishes and/or coverslips were allowed to dry up

while left under the tissue culture hood. We found out that if the coverslips were not

allowed to dry up completely the DRG neurons would not attach.

There is a very high correlation between DRG neurons survival and buffer temperature.

If isolation was done at 37oC, there was a high neuronal mortality by the second day in

culture. This effect was not seen when we used CSF medium at 4oC during the

isolation. When using Full medium (Hibernate A as prepared below), we noticed a

further increase in the neuronal survival, the neurons also displayed an improved

morphology, the dissociation was better and easier, and the dissociated cells displayed

a higher adherence. Therefore, the isolation was done by using Full Medium (FM =

3.1.1.2. Media and reagents for the isolation and culture of neurons

1. Cold Cerebrospinal Fluid (CSF): in 1L of dH2O add NaCl (7.36g), KCl (0.22 g),

KH2PO4 (0.163g), CaCl2 (0.266g), C6H12O6 (glucose 2.7g), Hepes (0.5g),

NaHCO3 (2.18g), Dextran (30g), and MgSO4 (0.32g).

2. DMEM: Dulbecco’s Modified Eagle Medium (DMEM) (2.2 mM, GIBCO cat:

11095) for a final volume of 500 ml add 10 % heat-inactivated fetal bovine serum

(GIBCO 1600-044), 5.5 ml of penicillin and streptomycin (GIBCO 15140-122),

and 5.56 ml Glutamax (GIBCO 35050) .

3. Hibernate A: Hibernate A Phenol red (Brainbits HA-pr. GIBCO, Springfield Il.) Up

to 300 ml; 6ml of B-27 supplement (GIBCO, cat: 17504-044, Grand Island

NY.USA); 0.75 ml of Glutamax 0.5 mM; 3 ml of penicillin and streptomycin.

4. Neurobasal: Basic Neurobasal (GIBCO 1X cat. 21103049) up to 400 ml, 8 ml of

B-27 2%; 1 ml of Glutamax 0.5 mM; 4ml of Penicillin-Streptomycin.

5. NGF: Nerve Growth Factor 2.5S, from Chemicon International, Temecula, CA.

6. PGE2: we started using the PGE2 from Calbiochem, Cat: 538904, San Diego CA.

and then we switched for more stable PGE2 (9-doxy-9-methylene-16, 16-dimethyl

prostaglandin E2) from Cayman chemical, Ann Arbor, Michigan.

7. Collagenase type I (400U/ml, Gibco-Invitrogen, Carlsbad, CA).

8. Trypsin 1:250,from Sigma Aldrich, St. Louis MO.

9. Optiprep TM (Axis-Shield, Oslo Norway).

The following gradient was used in order to get less myelin and more DRGs:

Optiprep pure was taken straight out of the bottle. Optiprep stock was prepared as

follows: for 10ml, 4.95ml pure optiprep + 5.05 ml hibernate A medium as stock, see

Color of Gradient

Percent of Gradient

For 1ml Volume For 8ml Volume

Red 12% 0.20mL opt pure + 0.80 mL HA

medium

1.6mL opt pure +6.4mL HA medium

White 9% 0.15mL opt pure + 0.85 mL HA

medium

1.2mL opt pure + 6.8mL HA medium

Red 7.4% 0.25mL opt Stock + 0.75mL

HA medium

2mL opt Stock + 6mL of HA medium

White 6% 0.20mL opt Stock + 0.80 mL

HA medium

1.6mL opt Stock + 6.4mL HA medium

Red 4.8% 0.15mL opt Stock + 0.85mL

HA medium

1.2mL opt stock + 6.8mL HA medium

Table 3. Values for pure Optiprep, prepared Optiprep and hibernate A to do the mixture for the gradient.

3.1.2. Animal Procedure

All the procedures were done conformed to New York University School of Medicine

Institutional Animal Care and Use Committee (IACUC). The experiments were done in

Sprague-Dawley rats that weighed around 270 g to 370 g, and 60 to 70 days old. The

rats were anesthetized using a cocktail of Ketamine and Xylazine getting a final

concentration of 80 mg/Kg of Ketamine and 12 mg/Kg of Xylazine. The cocktail was

injected intraperitoneal, allowing the animal to be anesthetized. Once the animal got

anesthetized, it was perfused through the aorta with the oxygenated cold artificial

cerebrospinal fluid (ACSF). The heart was reached cutting from the bottom of the

sternum along the fur and the skin over the diaphragm and through the ribs upwards

until the heart was seen. The heart’s apex and the right atrium were cut to let the blood

and the ACSF run through the whole circulatory system from the left ventricle to the

CSF run through, the animal was decapitated. Sometimes the brain was taken in order

to get the whole protein as a control for the western blots.

The animal’s skin was cut from the neck to the tail to let the vertebral column be seen.

A long cut of 4 cm from the bottom of the tail and side by side of the vertebral column

was made to let the bone scissors reach the vertebral column to be cut. Then the

vertebral foramina was gotten to put a 16 ½ in needle inside and did pressure with 10ml

of CSF upwards in a syringe, letting the spinal cord came out from cervical region by

hydraulic extrusion. All the muscles and tissues around the cervical and dorsal

backbone were cut in three pieces, and were put it in a 50 ml conical tube with full cold

medium (hibernate A prepared). All the time the samples were kept on ice. All that

tissue which was still on the dorsal, ventral and lateral sides of the backbone was taken

away on a glass petridish and under the stereoscope. Cutting on each side of the dorsal

spinous processes, exposing the roots and the ganglion made using small scissors a

window along the spinal canal of each backbone section. With sharp forceps, all the

ganglia were pulled out and their roots were cut, to reduce the amount of myelin in the

cultures. It is very important to cut the roots from the ganglia before starting the

enzymatic dissociation process. As the roots were cut the ganglia were collected into a

35 mm plastic petridish with FCM. After getting rid of all of the roots, the ganglia were

transferred to a 15 ml plastic conical tube and incubated at 37oC for 90 min in full medium containing 400U/ml of collagenase type I (GIBCO, cat. 17100-017), and 2.5

mg/ml of Trypsin (Sigma, Trypsin 1:250 from porcine pancreas. Cat: T-4799).

3.1.3. Dorsal Root Ganglia Neuron’s culture

After the 90 min of incubation were completed, the sample was centrifuged at 2556 g

(Beckman J6-HC rotor JS-4.2) for 5 min at room temperature, and then 6 ml of medium

were removed from the tube, the pellet which was the ganglia after the enzymatic

reaction was resuspended using gentle mechanical dissociation with sterile glass

Pasteur pipettes at different bores. 10ml of solution was completed using Hibernate A in

order to stop by dilution the enzymatic reaction. Once the enzymatic reaction was

centrifuge, after that 6 ml of media were removed from the tube, leaving a remain of 4

ml of medium with the pellet. This pellet becomes into the mixture of myelin and the

Dorsal Root Ganglia single Neurons. This pellet was resuspended and added carefully

in the top of a different concentration gradient column built using Optiprep (from

Optiprep TM, Axis-shield No. 1114542) plus Hibernate A solution at the following

concentrations: 4.8%, 6%, 7.4%, 9% and 12%. The entire column and the cell

suspension were centrifuged for 15min at 820 g at 4oC. When the time was finished the myelin and the cells were retained on each of the concentrations. The first two layers

4.8%, and 6%, were full of myelin and debris, so they were discarded. The layers 7.4%,

9% and 12% were plenty of DRG neurons and were put in a new vial but the pellet was

discarded also as a source of contamination for the culture. 7.4%, 9% and 12% layers

were mixed, in order to get cleaned DRG cells with Hibernate A up to 10 ml. This new

suspension was centrifuged for 5 min at 820 g at 4oC; the supernatant was discarded, getting the pellet and resuspending it in a certain volume of Neurobasal medium. Using

the Neubauer chamber, the cells were counted and Plated by 10 µl of suspension on

each PDL covered coverslips in the wells. To count live and death DRG neurons, 10 µl

of cell suspension plus 10 µl of trypan blue were used.

Once the cells were counted, they were placed in a multiwell of 24 well tissue culture

Plate, using PDL pre-coated coverslips, dividing the plate in two halves of 3 wells each,

one half for positive immunostaining samples (with primary antibody) and the other half

for the negative samples (without primary antibody). Vertically, 4 treatments were

organized in columns of 6 wells, each column was label as follows: first Neurobasal

(NB); second, Nerve Growth Factor (NGF); third, Prostaglandin E2 (PGE2) and fourth,

NGF + PGE2 (N+P) (see Fig 6). The case was placed in the incubator with humidified

atmosphere 5% CO2 at 37oC for 1 hour, in order to allow the cells to attach. Then 500 µl

(per well) or 2000 µl (per petridish) of media with the respective treatments were added.

This time was recorded to control the time the cells completed the 24, and 48 hours.

Based on previous findings the PGE2 is very unstable then the culture was fed two

times a day. Every day cells must be fed as fallows: in the morning putting the PGE2

Figure 6. 24 multiwell for cell culture, using 13mm coverslips PDL precoated.

Based on the information from Von Banchet, GS. et al 2003, the concentration of the

three treatments was 10-6 M of PGE2, and 100 ng/ml of NGF. The doses were

administrated in a final volume of 500 µl per well and 2000 µl per petridish one hour

after the cells were plated. PGE2 doses were administrated two times a day, in the

morning at around 9:00 a.m. putting 1 µl of 500 µM of PGE2per well with this treatment,

and in the afternoon at the same hour of the first doses (except day 2), half of the media

was changed, getting the concentrations of the treatments in each well. Cells were fixed

as protocol shows at the same time they were fed for the first time after the 48 hours of

cultured.

3.2. Immunostaining

For the cell culture, after 48 hours of treatment, the cells were washed three times with

PBS and then fixed with 4% Paraformaldehyde (PFD) and stored at 4oC. The

Immunohistochemistry was prepared for a double staining; it means that two primary

antibodies were used to show the two receptors, the NMDA subunit NR2B (from Santa

Cruz sc-1469) and NK1 (from Calbiochem, Anti-Neurokinin-1 receptor 393-407) or the

The fixed cells were equilibrated at RT for 20 min and then washed with PBS four times

5 min each. To avoid paraformaldehyde self expression, the cells were quenched with

Glycine 100 mM per 10 min on the shaker. The cells were incubated with 400 µl, 0.3%

Triton X-100 in PBS during 25 min at RT and then washed twice with PBS 1X for 3 min.

Cells were blocked 1 hour at RT with 10 % Normal Donkey Serum (NDS From

Sigma-Aldrich, D9663) prepared in PBS, 300 µl per well. At the end of the blocking time the

buffer was changed for the primary antibody and it was incubated overnight (O/N). The

primary antibodies were mixed in the same solution at its corresponding concentration.

The next day the primary antibodies were washed 4 times for 5 minutes with 1X PBS

and incubated with the secondary antibody (Alexa-Fluor 546 Donkey anti goat from

Molecular probes) diluted in PBS for 1 hour at RT. Then incubate the second secondary

antibody against NK1r (Alexa-Fluor 488 goat anti-rabbit, from Molecular probes) for one

hour, followed by three times wash during five minutes each. Then the cell’s nuclei were

stain with Hoechst (Bisbenzimide H 33342, from Sigma, product No. B2261), stain at

10µg/ml during 20 minutes. These coverslips were mounted using Aqua poly/Mount

(from Polysciences, Inc. cat # 18606). All the samples were kept in darkness in order to

avoid bleaching the staining.

3.3. Western Blot Protocol

3.3.1. Protein EXTRACTION FROM CELL CULTURE

Some experiments were done with the same amount of cells used in the

immunostaining trying to get the bands of 178 kDa for NMDAr (NR2B) and 75 kDa for

NK1r, but it was not enough. We decided to increase the amount of cells using more

ganglia. An average of ganglia obtained was 44 and an amount of 252,000 cells plated

per petridish PDL coated. All the treatments were placed during the 48 hours as in the

cell culture.

Once the two days of culture were completed, all the treatments (NB, NGF, PGE2 and

NGF plus PGE2) were put on ice and washed two times with 1000 µl of PBS (Invitrogen)

plus Protein Inhibitors Cocktail (PIC) 1:200, DPBS +Ca+2, + Mg+2 five minutes, then

-Ca+2,- Mg+2for five minutes. Then 60 µl of Ripa (from Millipore, cat # 20-188) enriched

and left on ice for 30 min. Meanwhile, all the cells were collected into an eppendorf, and

then vortex for 1 min. After the time all the vials were centrifugated at 4oC for 15 min at 17000 g. 10 µl from each treatment were aliquot for protein determination and the

sample stored at -80oC.

3.3.2. PROTEIN DETERMINATION

To determine the protein concentration from the cell cultured under different treatments

the Lowry assay was made under the following protocol:

The system was placed using three rows of small test tubes one for the protein control,

and two more rows for samples row 2 and 3. Dilutions were made in the first row and

then used for the standardization curve using BSA controls (2µg/ml). The control was

prepared adding 1 ml of ddH2O to each of the tubes in Row 1. Then, for controls, the

amount of ddH2O in the following table (table No. 3) was taken out and added back the

same amount of BSA stock solution (2mg/mL in H2O, 0.7mL total volume using either

Bio-Rad Standard Protein II).

[BSA] - ddH2O in

µLs

+ BSA in µLs

0 µg/ml 0 0

200 µg/ml 10 10

400 µg/ml 20 20

600 µg/ml 30 30

800 µg/ml 40 40

1000 µg/ml 50 50