SURGICAL DESCRIPTIONS
ADRENAL DEMEDULLATION:
The animal will be anesthetized. A dorsal cut in the skin will be made just above the
kidney (just posterior to the last rib and lateral to the midline). A rat-toothed forceps will
be used to pull the fat surrounding the adrenal gland out of the incision. Care will be
taken to avoid grabbing the adrenal gland with the forceps. Once the adrenal gland is
exposed, a 1mm incision in the adrenal cortex, distal to the vascular input will be made
with a #11 scalpel blade and the gland will be gently compressed such that the adrenal
medulla is expressed through the incision according to the method of Tonge and Oatley
(Physiol. Behav. 10:497-505, 1973). The muscle will be sutured with sterile absorbable
sutures and the skin will be closed with sterile wound clips.
AUDITORY RECORDING:
The animal will be anesthetized. A skin incision will be made over the brain area of
interest. Any bone overlying the area will be removed with a scalpel blade or small
rongeurs. Lesions or manipulation of the relevant brain tissue or sensory organ will be
made with a small heat cautery. The skull will regenerate and thus no effort will be made
to replace the removed section. Skin will be sutured with 6-0 Prolene* or surgical glue
(VetBond) will be utilized.
COCHLEAR REMOVAL:
The animal will be anesthetized. A skin incision will be made behind the pinna to expose
the auditory bulla. A small hole will be made in the bulla, and the cochlea will be
removed by aspiration with an 18 gauge needle connected to a suction pump. The
procedure takes approximately 30 minutes. Confirmation of deafness will be done by
non-invasive auditory brainstem recordings after the animal reaches maturity and by
histological verification after euthanasia. The animals often sustain some damage to the
vestibular system as a result of the cochlear removal, and must be partially-hand fed
puppy milk replacer until they open their eyes at four weeks of age and are able to orient
by use of visual cues.
DECEREBRATION:
The animal will be anesthetized. A skin incision will be made over the skull and a
bilateral craniotomy will be performed by drilling burr holes into the parietal skull.
Subsequently, the portion of bone superior to the central sagittal sinus will be removed.
The dura mater will be breached and reflected. Decerebration will then occur in which
brain tissue at the intercollicular level will be slowly transected using a dull spatula.
Because the spatula does not have a sharp edge, it does not cut through arteries,
diminishing the likelihood of bleeding. Transection will be confirmed visually by a clear
view of tissue separation. In case such a clear view cannot be achieved, a fraction of the
brainstem tissue (0.2-0.3mm) will be removed after another transection immediately
below the inferior colliculus. The completion of decerebration will be determined by
visual inspection of the brain tissue with respect to the complete separation of the
brainstem from the upper levels of brain tissues.
Once the animal is decerebrated, the animal will no longer be able to detect a pain
sensation. At this time, the anesthesia will be terminated and the animal will be
maintained on a paralytic agent such that it will be unable to move. At the termination of
the experimental procedures, the animal will be euthanized without recovery from the
influence of the paralytic.
SURGICAL DENERVATION OF ADIPOSE TISSUE:
The animal will be anesthetized. The incision site for the denervation depends on the fat
pad to be denervated. A small skin incision will be made. The epididymal white adipose
tissue (WAT) denervation requires a ventral incision that invades the peritoneal cavity.
The inguinal WAT denervation requires only a ventral incision along the rear dorsal
flank, as this is a subcutaneous fat pad. The retroperitoneal WAT denervation requires a
dorsal incision slightly more anterior to the inguinal cut, but invades the peritoneal cavity
as this is an internally located WAT pad. The nerves will be cut with microscissors with
the aid of a dissecting microscope as they enter the fat pads. The skin will be closed with
sterile absorbable sutures and the incision will be closed with wound clips.
CHEMICAL DENERVATION OF ADIPOST TISSUE (6 OHDA):
The animal will be anesthetized. We will sympathetically denervate white adipose tissue
(WAT) using local injections of the catecholamine neurotoxin 6-hydroxy-dopamine
(6OHDA). The incision site for the denervation depends on the fat pad to be denervated.
A small skin incision will be made. The epididymal white adipost tissue (WAT)
denervation requires a ventral incision that invades the peritoneal cavity. The inguinal
WAT denervation requires only a ventral incision along the rear dorsal flank, as this is a
subcutaneous fat pad. The retroperitoneal WAT denervation requires a dorsal incision
slightly more anterior to the inguinal cut, but invades the peritoneal cavity as this is an
internally located WAT pad. The WAT or BAT pad will be injected with micro amounts
and volumes of the 6OHDA (Sigma Chemical, St. Louis, MO; 10 injections of 1
microliter of 4mg/100 microliter 6OHDA in sterile saline per fat pad). The incision will
be closed with wound clips. In some cases where we are testing for overall effects of
denervation on systems physiology and/or behavior, we use a between animals design so
this will be done bilaterally with a separate group receiving saline control injections. In
other cases, we are looking at the effects of denervation on a fat pad response to be
contrasted with the response on the opposite side. In this case the contralateral fat pad
receives similar injections of the saline vehicle. As these are relatively small volumes in
such large target tissues, the toxin does not escape into neighboring tissues. Note that
6OHDA is not an IBC regulated toxin and is safe to handle. We do, however use the
same precautions as with real toxins: 1) injections will be done in a safety hood with the
individual performing the injections wearing gloves, mask, lab coat and goggles, and 2)
all materials will be autoclaved after use.
CHEMICAL DENERVATION OF ADIPOST TISSUE (Capsaicin):
The animal will be anesthetized. We will sympathetically denervate adipose tissue using
local injections of capsaicin, the pungent part of red chili peppers. Capsaicin kills small
unmyelinated sensory neurons. Although many have injected capsaicin systemically to
produce a global peripheral sensory denervation, we will use it to produce a local
selective sensory denervation of white adipose tissue (WAT) or of brown adipose tissue
(BAT). The incision site for the denervation depends on the fat pad to be denervated. A
small skin incision will be made. The epididymal WAT denervation requires a ventral
incision that invades the peritoneal cavity. The inguinal WAT denervation requires only
a ventral incision along the rear dorsal flank, as this is a subcutaneous fat pad. The
retroperitoneal WAT denervation requires a dorsal incision slightly more anterior to the
inguinal cut, but invades the peritoneal cavity as this is an internally located WAT pad.
The pad will be visualized and the injections will be made to cover the pad. Capsaicin
(Sigma) will be injected locally as 20 microinjections (2 microliters per injection, 40ul
total) of 200 micrograms/microliter of capsaicin dissolved in a sterile vehicle consisting
of 10% of ethanol, 10% of Tween 80 and 80% of 0.9% NaCl using a microsyringe. The
incision will be closed with sterile wound clips. In some cases where we are testing for
overall effects of denervation on systems physiology and/or behavior, we use a between
animals design so this will be done bilaterally with a separate group receiving saline
control injections. In other cases, we are looking at the effects of denervation on a fat
pad response to be contrasted with the response on the opposite side. In this case the
contralateral fat pad will receive similar injections of the saline vehicle. Note that
capsaicin will be used to alleviate pain. Also note that capsaicin is being injected directly
into tissue and will be done in such a way as to minimize if not prevent leakage outside of
the tissue. Capsaicin also does not require IBC approval. The experimenter, however,
will mix the solution in a biological safety cabinet and will also don gloves, a lab coat,
and eye protection.
ELECTROPHYSIOLOGY:
The animal will be anesthetized. This procedure will be conducted to explore the
response of individual neurons in the auditory cortex to auditory or visual stimulation.
The head will be stabilized in a stereotaxic head holder. The animal will be paralyzed
with gallamine triethiodide (to eliminate eye movements) if necessary (this is not
expected) but at any rate will be artificially respired at a rate of 30-40/min. In the case of
paralysis, withdrawal reflexes cannot be used as an indication of pain and heart rate will
be used to assess the depth of anesthesia. The cortex will then be exposed unilaterally by
making a C-shaped incision in the scalp and performing a craniotomy and durotomy over
the region of interest. The brain tissue will be protected throughout the procedure by
covering it with warm mineral oil or 1% agar in saline.
Extracellular multiunit recordings will be made with tungsten microelectrodes. By
making tracer injections before or during the electrophysiology, the physiology can be
directly correlated with the connectivity, reducing the number of animals needed. The
bladder will be emptied at intervals by gentle manual pressure so that the animal remains
clean and dry. Auditory stimuli stereo headphones connected to earplugs will be used for
presentation of closedfield auditory stimulation. Visual stimuli to aid in locating visual
units, both visual searching stimuli and weak, 1 Hz electrical stimulation of the optic
chiasm will be used. The stimulation does not cause any damage. The nictitating
membrane of each eye will be retracted with phenylephrine, the pupils will be dilated
with atropine sulfate, and contact lenses will be used to focus the animal’s eyes on a
tangent screen as confirmed by retinoscopy. Visual stimuli will be presented on a screen
in front of the animal.
For an analysis of the role of inhibitory circuitry in visual response properties, we will
employ in vivo iontophoretic application of drugs. A tungsten-in-glass microelectrode
will be glued onto a three or five-barreled micropipette with physiologically-relevant
concentrations of GABA or glutamate agonists and antagonists in the barrels. We will
measure the current threshold to the iontophoretically-applied drugs, increasing the
current in 20 nA steps. The current injections are not of sufficient magnitude to damage
the brain tissue. Euthanasia via perfusion through the heart (saline followed by aldehyde
fixatives) will follow this terminal electrophysiological recording session, which can last
up to 120 hours. The brain will then be extracted for histological analysis.
ENUCLEATION:
The animal will be anesthetized. This manipulation eliminates the trophic and neural
activity-based contribution of the sense organs to brain development. Ferrets do not open
their eyes until one month of age thus the procedure will be done prior to P30. The
animal will be placed supine with an eye uppermost. The surgical site will be cleaned
with betadine and draped (note we are not concerned with damage to the eye caused by
betadine since the eye is being removed). The lid will be opened along the future eyelid
margin (visible at this age), and the orbit will be separated from the surrounding
conjunctiva with a microscissor. The orbit will be removed in its entirety, taking care to
eliminate all pigmented epithelium that could conceivably regenerate some
photoreceptors. There is typically no bleeding associated with this procedure even though
the vessels are not ligated, clamped, or cauterized, but to prevent any possible loss of
blood, the orbit cavity will be packed with Gelfoam. The eyelids will then be sutured (6-0
silk) or glued (VetBond) closed. It is not critical in this case to ensure that there are no
light leaks through the suture line, because the eyes are absent. The goal is to protect the
orbit cavity from dessication or contamination. Monocular manipulations cannot be used
because there is cross-talk between the two brain hemispheres at the thalamic and cortical
levels. Optic nerve section is less reliable given the possibility that the retinal fibers could
regenerate. Blind animals adjust quickly to their surroundings, and this is particularly true
of crepuscular, burrowing animals such as ferrets. There will be no suffering or pain
resulting, but it will be requested of the animal care personnel that the compass
positioning of the cage and items in the cage be as constant as possible in order to
facilitate orientation within the home cage.
EYELID SUTURE:
The animal will be anesthetized. Ferrets do not open their eyes until one month of age
thus the procedure will be done prior to P30. Lid suture prevents form vision but not light
detection, and thus has different consequences for cortical development than does
enucleation or dark-rearing. The procedures are substantially the same except that the
orbit will not be removed and more care must be taken to protect the delicate cornea. The
neonatal animal will be placed supine with an eye uppermost. The eye will be washed
with sterile saline and coated with petroleum jelly for protection from betadine wash. The
area around the eye and lids will then be cleaned with betadine and draped. The lid will
be opened along the future eyelid margin (visible at this age), and the eyelid margins will
be trimmed with a microscissor, removing just the thickened layer of cells along the
margins, i.e. as little as possible to create a clean surface for apposition. The margins will
be carefully sutured such that the surfaces are in close apposition, promoting permanent
fusion of the lids. The eyelids will be sutured together with closely spaced 6-0 silk
stitches such that they are completely closed. Care will be taken to ensure that the
sutures placed in the eyelids do not penetrate the full-thickness of the eyelid so as to
prevent the sutures scractching the underlying eye. It will be critical in this case to ensure
that there are no light leaks through the suture line, and thus the suture line will be
visually inspected every 30 min for 3 hours and then daily thereafter for 20 days.
GONADECTOMY:
1) Castration Using Scrotal method:
The animal will be anesthetized. The animal will be placed in dorsal recumbency
following surgical preparation. The skin of the scrotum will be cleaned of all fecal
material and surgically prepared consistent with the GSU surgical patient preparation
policy. A small median incision of about 1 cm will be made through the skin at the tip of
the scrotum. At this point, blunt dissection will be used to exteriorize the testicle while
still inside the parietal vaginal tunic (commonly referred to as a "closed" castration). A
ligature with absorbable suture will be placed around the parietal vaginal tunic proximal
to the testicle and the testicle will be removed by cutting the parietal vaginal tunic distal
to the ligature. Should the parietal vaginal tunic be inadvertantly opened during the
procedure (commonly referred to as an "open" castration) then a ligature with absorbable
suture will be placed around the vas deferens and the blood vessels and then cut distal to
the suture, allowing removal of the testitle. (cautery of the vas deferens and blood vessels
is also allowable instead of suture). In the case of the open castration, a final ligature will
be then placed around the parietal vaginal tunic. The scrotal incision will be closed with
wound clips.
2) Castration Using Abdominal method:
The animal will be anesthetized. The animal will be placed in dorsal recumbency
following surgical preparation. The abdominal wall will be incised. The testicle will be
located and retracted through the abdominal incision by gently grasping the peritesticular fat tissue located in the caudal abdomen on each respective side. The vas
deferens and testicular vessels will be cauterized or ligated with absorbable suture. The
vas deferens and testicular vessels will be transected distal to the ligature or site of
cautery. The abdominal wall will be closed with an absorbable suture in a simple
interrupted pattern and the skin incision will be closed with wound clips.
INTRACEREBRAL MICROINJECTION:
The animal will be anesthetized. Discrete microinjections of substances into the brain
can permit the experimenter to stimulate or inhibit behaviors and/or physiological
responses. A small single incision will be made on the midline on top of the head to
reveal the underlying bone fissures, the position of the cannulae will be determined based
on experience (e.g. no stereotaxic atlas for Siberian hamsters) or, as it relates to other
species, detrmined via the use of a stereotaxic atlas. A hole through the skull will be
trephined to allow insertion of the guide cannulae to the appropriate depth. The cannulae
will be anchored to the skull with strategically placed jeweler’s screws, cyanoacrylate
ester glue base and then dental acrylic. The incision will be closed with wound clips
being careful not to impair the eyelids. Microinjections will be made through an infusion
cannula that penetrates past the tip of the guide cannula. Between injections, a sterile
obturator will be inserted in the guide cannulae.
LIPECTOMY:
The animal will be anesthetized. An abdominal incision will be made for epididymal or
retroperitoneal white adipose tissue removal, whereas a dorsal subcutaneous incision will
be made for inguinal or dorsosubcutaneous white adipose tissue removal. Depending
upon the intent of the experiment, one of the pair of these fat pads will be removed (e.g.,
one inguinal), both pads of the pair will be removed (e.g., both epididymal), or
combinations of single and double removal will occur (one inguinal, two epididymal etc),
the latter creating a “dose-effect” for fat removal. The fat pads will be removed by blunt
dissection to minimize bleeding with care taken to minimize disruption of the blood flow
and trauma to the adjacent tissues (testes for epididymal WAT). Sham lipectomy only
differs from the real surgery in that the pads will not be removed in the former.
Depending upon the site of removal, the muscle incision will be closed with sterile suture
(intraperitoneal: epididymal and retroperitoneal WAT) and the skin incision will be
closed with wound clips (all pad removals).
NERVE TRACT TRACER:
The animal will be anesthetized. In order to determine which brain areas are connected
to one another, as well as the nerves that innervate peripheral tissues, various chemical
dyes (or viruses) will be injected into the brain or peripheral nervous system target tissue
and they will either be taken up by the neurons at their distal end and transported to the
cell body (retrograde tract tracers), or taken up by the cell body and transported to the
proximal end of the neuron (anterograde tract tracers). In either case, the tracers will only
label one neuron in the chain of neurons making up a neural circuit.
Peripheral injection of nerve tract tracers: Animals will be anesthetized and placed on a
sterile pad for injection into a peripheral tissue or into the sympathetic chain. An incision
will be made at the site of injection. The retrograde tract tracers horseradish peroxidase,
rhodamine-labeled microspheres or FluoroGold will be microinjected into the peripheral
target to label the sympathetic chain. Peripheral targets will include the adrenal medulla,
white adipose tissue (epididymal, retroperitoneal, inguinal, dorsosubcutaneous,
perirenal), or brown adipose tissue (interscapular). To obtain bi-directional confirmation
of the neurons in these circuits, the anterograde tract tracers DiI, dextran amines or
Phaseolus vulgaris leuccoaglutinin (PHAL) will be injected into the sympathetic chain to
label the neurons going to the target tissues (all those listed above) for anterograde
tracing in separate animals. For all tracers 1-2 microliters or less of tracer will be injected
into the peripheral tissue. Concentrations of each vary according to the preparations as
purchased from a variety of suppliers. For epididymal and retroperitoneal white adipose
tissue (WAT) and the adrenal medulla, an off-centered midline incision will be made in
the abdominal area and the target identified and injected. For the inguinal and
dorsosubcutaneous WAT pads, the tissues are exposed by incisions located over the
bottom and top leg areas respectively. For interscapular brown adipose tissue (BAT), the
area over the pad on the dorsum of the animal between the scapulae will be incised to
expose the pad. The tracers are pressure-injected directly into the target tissues using a 1
or 0.5 microliter syringe -- anterograde (sympathetic chain) or retrograde (WAT, BAT,
adrenal medulla). The muscle will be sutured shut with sterile sutures, and the overlying
skin will be closed with wound clips.
Brain injection of nerve tract tracers: After anesthetization, the animal will be mounted
into the stereotaxic apparatus and a small incision will be made on the midline on top of
the head to reveal the underlying bone fissures. The position of the cannulae will be
determined based on experience (no stereotaxic atlas for Siberian hamsters) and a hole
trephined to allow stereotaxic lowering of the injection cannulae (30 gauge) to the
appropriate depth (usually ~0.5 mm above the desired site). The pressure injection (100500 nl depending upon the site) will be made and the cannula will be held in place for 30
sec to 1 min to avoid reflux up the outside of the cannulae. The cannula will be raised, the
skull hole will be covered with sterile gel foam, and the incision will be closed with
wound clips.
Defining neural circuits using viral transneuronal tract tracers: In order to trace an
entire circuit in the same animal, a transneuronal viral tract tracer will be required and we
have been using the attenuated strain (Bartha*s K strain) of the pseudorabies virus (PRV)
to map the functional connections from white adipose tissue (WAT), brown adipose
tissue (BAT) and the adrenal medulla to the brain. Note that we will be using a severely
attenuated Bartha*s K strain of the PRV, the Bartha*s K strain * a strain that is so
attenuated that it is used to vaccinate swine against pseudorabies. Also, note that there are
no documented cases of PRV Bartha*s K strain infections in humans, only the wild-type
PRV which we do not use. The virus also cannot be transmitted from animal to animal
through the air (years ago we did several tests where one animal was injected with PRV
in a cage of animals and none of the other animals became infected). The low virulence
of this PRV strain also is apparent in that ~25-35% of animals we directly inject with the
virus do not become infected. As precautionary measures, the procedure will be done
under a fume hood, the inside top of which will be covered with bench cloth. The animal
will reside on a sterile pad placed within a stainless steel pan and the vial of virus will be
on ice and also within a stainless steel pan, both done to contain any accidental virus
spill. An incision will be made to expose the tissue of choice. The injections will be
made by pressure injection (5-15 loci within the tissue depending upon its size; typically
viral titer ~1-3x108 plaque forming units, each injection ~125 nl). Sometimes we will mix
cholera toxin B subunit (CTb) with PRV because CTb is a very sensitive retrograde tracer
(PRV is retrograde too), but unlike PRV, it only fills the first neurons it comes in contact
with and not the next neurons in the circuit. Therefore, it is useful to tell us which of the
PRV-infected neurons in the circuit are the first ones labeled (last neurons in the circuit
because the circuit is labeled backwards [i.e., retrogradely]). The CTb has had the
contaminating endotoxin removed and does not have a MSDS because the manufacturer
(List Labs) has demonstrated it is not dangerous as would be the case if it was cholera
toxin itself. Each injection will be across a 1 min period with 1 min where the injection
needle remains in place to minimize reflux out of the site. The tissues to be injected
include WAT (epididymal, retroperitoneal, inguinal, dorsosubcutaneous, perirenal), or
BAT (interscapular) or the adrenal medulla. The surgical incision will be closed with
sterile sutures (muscle) and sterile wound clips (skin). We also are interested in using the
power of the PRV to label circuits within the brain itself, rather than starting in the
periphery as above. In this case the protocol will be exactly as for the injection of a toxin
into the brain except instead of injecting the toxin, we inject the PRV. Because the virus
does not have to travel very far in the brain compared with injections in the periphery, the
post-injection time will be much shorter (8-24h maximum). As above, the PRV may be
combined with CTb for the same reasons. We will use isogenic PRV strains of the
Bartha's K strain that have been genetically engineered to produce different reporters
(fluorescent red or green, beta galactosidase) or the standard non-wild-type strain. The
virus chosen depends on our experimental design and intentions (fluorescent for confocal
microscopy; standard strain or beta galactosidase for light microscopy). For double-virus
studies, a second injection in a different target tissue will be used. The injections will be
done in a Biological Safety Cabinet and, despite the safe nature of this attenuated virus
that does not infection humans and frequently does not infect the animals that receive
direct injections of this virus used to immunize swine against wild-type pseudorabies
virus, the person injecting the virus will don gloves, goggles, a lab coat and eye
protection. All materials used in the injection will be subsequently autoclaved as will be
the bedding and cages of the injected animals.
Sensory viral tract tracing with H129 strain of Herpes Simplex Virus 1: We wish to
microinject the Herpes simplex virus type 1 (H129 strain) into white adipose tissue
(WAT) and perhaps brown adipose tissue (BAT). The injection procedure will be almost
identical to that used for PRV described above except that this will be done under the
more restrictive and safety precaution measures of BSL-2. Therefore, the person injecting
the virus will don gloves, goggles, a lab coat, and eye protection. All materials used in the
injection will be subsequently autoclaved as will be the bedding and cages of the injected
animals. Injections will be done in a Biological Safety Cabinet, the inside top of which
will be covered with bench cloth. The animal will reside on a sterile pad placed within a
stainless steel pan and the vial of virus will be on ice and also within a stainless steel pan,
both done to contain any accidental virus spill. We will inject epididymal, retroperitoneal,
inguinal or dorsosubcutaneous WAT or interscapular BAT (with one injection site per
animal). Incisions are made and microinjections of H129 (viral titer = 1 x 1010 plaqueforming units per ml) will be made via a Hamilton microsyringe with a sharpened
beveled tip. Depending upon the size of the fat pad, differing numbers of 100-500 nl
injections will be given to cover the extent of the pad. For the largest pad (inguinal
WAT), it will require 10 injections. Each injection will be given across a 1 min period
and then the needle will be held in place for an additional minute to minimize reflux.
Depending upon the WAT pad location, the surgical incision will be closed with sterile
sutures (muscle for internally located pads e.g. retroperitoneal, epididymal) followed by
wound clips (skin) and only wound clips for the subcutaneous pads (inguinal and
interscapular BAT).
OOCYTE COLLECTION:
The animal will be anesthetized. A few lobes of ovaries will be removed after a small
abdominal incision (~5 mm). The surgical incision will be closed using absorbable
sutures internally and non-absorbable sutures on the skin and the frogs are allowed to
recover from the anesthesia. The frogs will then be returned to the animal housing
facility. Antibiotics will not be given to the animals as the frog skin secretes antibiotic
peptides that protect the wound from infection.
OSMOTIC MINIPUMP IMPLANTATION:
Subcutaneous infusion: The animal will be anesthetized. An incision will be made in the
skin, the sterile minipump will be inserted subcutaneously, and the incision will be closed
with wound clips. This may have to be repeated for long duration experiments to replace
old pumps with new ones.
Infusion into a brain cannula: The animal will be anesthetized. An incision will be
made in the skin, the sterile minipump will be inserted subcutaneously, and the pump will
be connected via sterile tubing tunneled under the skin to a head cannula (surgical
placement of the head cannula described elsewhere). The incision will be closed with
wound clips. This may have to be repeated for long duration experiments to replace old
pumps with new ones.
Infusion into a vessel: The animal will be anesthetized. An incision will be made in the
skin, the sterile minipump will be inserted subcutaneously, and the pump will be
connected via sterile tubing tunneled under the skin to a cannulated vessel (surgical
placement of the vessel cannula described elsewhere). The incision will be closed with
woundclips. This may have to be repeated for long duration experiments to replace old
pumps with new ones.
OVARIECTOMY:
The animal will be anesthetized. The animal will be placed in ventral recumbency after
being surgically prepared. A small incision will be made in the skin approximately half
way between the shoulder-blades and the base of the tail and the skin will be bluntly
dissected away from the muscle to allow for movement to either side laterally.
Alternatively, bilateral incisions will be made in each flank. A muscle incision will be
made about 2/3 of the way down the side of the body. The ovary, surrounded by fat
should be directly underneath the incision. One other alternative will be to make a
ventral midline incision with the animal positioned in dorsal recumbency. In all cases,
the ovary will be pulled out through the incision by grasping the periovarian fat. Care
will be taken to avoid touching the ovary itself. Fat and blood vessels will be dissected
away from the junction of the Fallopian tube and the uterine horn and the junction will be
cut. A ligature will not typically be needed but may be applied with absorbable suture at
the proximal end of the uterine horn and fallopian transected. In lieu of a ligature, cautery
may also be utilized. The muscle incision will be closed with absorbable suture. Wound
clips will be applied to close the skin incision.
PERFUSION:
The animal will be anesthetized. The animal will be placed in dorsal recumbency. The
hair will be wet down with alcohol (or hair will be clipped) before making an incision to
expose the thoracic cavity. A (blunted) needle will be inserted into the left ventricle and a
small incision will be made in the right atrium. The perfusion pump will be started
(generally starting with saline). When the liver has blanched, the switch will be made
from saline to the fixative. Sometimes the descending aorta will be clamped distal to the
liver to concentrate perfusion in the upper body.
PINEALECTOMY:
The animal will be anesthetized. In order to test whether a given seasonal response is
mediated via the pineal and its principle hormone, melatonin, the pineal gland will be
removed (pinealectomy). An incision will be made over the skull and a trephine will be
used to make a hole in the skull above the confluence of the sinuses. The sinus will be
penetrated with a fine-toothed forceps to remove the pineal gland. Bleeding will be
stopped by inserting a gelatin pellet into the trephined hole. The incision will be closed
with sterile wound clips.
SILASTIC CAPSULE IMPLANTATION:
The animal will be anesthetized. At the time of ovariectomy or castration, hormone
replacement via Silastic capsules (5-30 mm long constructed of 0.062 in internal
diameter, 0.125 outside diameter; sealed with with Medical Silicone Type A (Dow
Corning)) containing either crystalline cholesterol (control), estradiol, testosterone or
dihydrotestosterone (androgen that is not aromatized to estradiol) will be implanted
subcutaneously near the back of the neck. The skin on the dorsal surface of the neck
(scapular region) will be incised with a 1/2 cm transverse incision. A subcutaneous
pocket will be created with a hemostat, the capsule will be inserted into the pocket and
the incision will be closed with wound clips. All capsules will be washed prior to
implantation to minimize transient high levels of hormone release. These capsules are
used to achieve stable plasma levels of the hormones within the physiological range.
VESSEL CANNULATION:
The animal will be anesthetized. The animal will be placed in dorsal recumbency. An
incision will be made in the skin on the ventral surface of the neck and blunt dissect
through the muscle will be conducted to expose the external jugular vein. At least 1 cm of
vein should will be typically exposed. A silk suture will be tied tightly at the anterior end
of the vein. A very loosely tied suture will be placed around the posterior end of the vein
(do not close the knot). A “catheter introducer” will be inserted into the vein between the
two sutures. The catheter (typically Silastic tubing) will be inserted into the vein and
advanced towards the heart. The posterior suture will be closed around the vein and
catheter to fix the catheter in place. Another suture will be placed around the vein and
catheter nearer the anterior end of the vein. The other end of the catheter will be tunneled
subcutaneously to exit the animal through a small skin incision on the dorsal surface of
the neck. The catheter will be encased in dental cement, and anchored to a square of
durable mesh for subcutaneous placement in the mid-scapular region to stabilize the
catheter and keep it from moving. Care will be taken to assure that there is enough
catheter length to allow for animal movement and growth so as not to dislodge the
catheter from the vein. The patency of the catheter will be checked by gently aspirating
blood from the vessel and then flushed with saline, followed by a heparine/saline (heplock). The end of the catheter will be capped with a stainless steel pin. The skin will be
closed with wound clips.