the basic mechanism of action in the freezing of tissue is the formation of ice crystals which results in the disruption of cell membrane and its organelles .
cells exposed to freezing temperatures of about 5 to 15  c at the periphery of the ice crystal / ice ball primarily undergo apoptotic cell death , whereas colder temperatures at the center of the cell leads to necrotic instead of apoptotic cell death ( jackson et al . 2005 ;
, necrotic cell death occurs at colder temperatures ( < 20  c ) because intracellular ice formation directly damages cell organelles and membranes . at the periphery where the temperatures are warmer ( 20 to 0  c )
the extracellular ice formation causes the environment to become hyperosmotic , thus removing water from the cells .
drastic osmotic changes within a   cell leads to severe cellular dysfunction resulting in apoptotic cell death ( pegg 2010 ) .
the local application of low temperatures has been used as a medical therapy for centuries before being adapted by modern medicine ( cooper and dawber 2001 ) .
early use of cold occurred over a small range of temperatures ( + 10 to 5  c ) and treatment resulted in the temporary inactivation of the nerve ( neuropraxia ) , which produced a nerve conduction block that could last from several hours to days ( rosenberg and heavner 1980 ; gage et al .
since then , cryosurgery has become the most common advanced use of cold for medical treatment .
though this method is well established and has been proven safe through decades of clinical use , it was not considered an ideal method for the treatment of motor or sensory nerves due to the extreme low temperatures used during treatment ( ~180  c ) . with new technological advancements ,
it has become possible to treat tissue over more specific temperature ranges , allowing for the treatment of sensory or motor nerves , known generically as cryoanalgesia and cryoneuromodulation , respectively .
focused cold therapy ( fct ) is the direct application of low temperatures to inhibit signaling of peripheral nerves .
exposure to temperatures between 20 and 88  c leads to axonal and myelin degeneration , also known as wallerian degeneration ( campos et al .
morphologically , this process is characterized by a beading appearance followed by granular disintegration of the axons at and distal to the site of exposure ( feng et al .
the acellular nerve structures such as the endoneurium , perineurium and epineurium are preserved , allowing normal axonal regeneration and remyelination ( moorjani et al .
this is in contrast to more traumatic nerve injuries ( i.e. , transection or thermal heat lesions ) , which disrupt the acellular nerve structures , sometimes causing neuroma formation and aberrant axon regeneration ( campos et al . 2009 ) .
previous studies have assessed the recovery of the peripheral nerves following cryogenic injury and detail the behavioral , electrophysiological and pathological recovery of these peripheral nerves after exposure to temperatures as low as 120  c ( barnard 1980 ; willenbring et al .
however , these studies focus on the structural and functional recovery of sensory nerves ; there have been very few studies performed to identify the effects of fct of peripheral motor nerves . in particular , there has been a lack of studies demonstrating long - term histological and physiological observations after multiple treatments .
fct can be used on motor nerves for various medical conditions , including but not limited to : movement disorders , muscle spasms , muscle hyperactivity and/or any condition where reduction in muscle movement is desired .
this paper will present a rat nerve model and demonstrate a temporary loss of motor function via the application of a cryotreatment on the sciatic nerve of the hind limb .
the physiological and histological changes up to 16  weeks after treatment will be presented .
animal studies were performed at stanford animal facilities ( palo alto , ca , usa ) , and the study protocol was approved by the stanford institutional animal care and use committee .
twenty - seven adult female sprague  dawley rats ( 250300  g ) were housed two to a cage with standard chow and water available ad libitum .
the myoscience cryo - touch ii device ( redwood city , ca , usa ) was used to apply fct to the nerves ( fig .  1 ) .
this handheld device delivers compressed liquid nitrous oxide to a close - ended 27 gauge , 6  mm long three needle probe ( trident probe ) .
a type t , bifilar 0.0015 thermocouple was soldered on the tip of a cryoprobe to monitor treatment temperatures.fig . 
1myoscience cryo - touch ii device myoscience cryo - touch ii device during the studies , animals were surgically treated with the device on the tibial nerve of the left leg .
the animals were placed under general anesthesia with a mixture of 2.5  l / min o2 and 2.5  % isoflurane . under aseptic conditions , the sciatic nerve was surgically accessed through the thigh targeting the trifurcation point ( tibial , sural and peroneal nerves ) .
the tibial nerve was visually identified and the cryoprobe placed in direct contact with the nerve .
the device applied a 1  min treatment at 56  c ( 55.9  c average ; range 51 to 61  c ) .
after treatment , the nerve was returned to the host bed and the incision was closed using standard two - layer technique .
the animal was allowed to recover from the anesthesia and then returned to its standard housing .
physiological toe spread and motor function were assessed twice a week until return to normal function was observed .
a modified toe - spread test was performed to determine the change in muscle function ( varejo et al .
toe spread was determined by lifting the rat by the base of the tail with the legs hanging freely .
the toe - spread reflex was observed with the following ratings : normal reflex with all toes spreading out received a score of 0 ; weak reflex with some toes partially spreading apart received a score of 1 ; and lack of reflex with all toes clubbed together received a score of 2 .
motor function was measured by trained laboratory personnel who were blinded to the animal study group .
these reviewers assessed the walking behavior of the animal and assigned a score ( brummett et al .
the motor function was observed with the following ratings : normal walking function received a score of 0 ; normal dorsiflexion ability while walking with curled toes received 1 ; moderate dorsiflexion ability while walking with curled toes received and no dorsiflexion ability while walking with curled toes received 3 .
the treated nerves were dissected at 2 , 8 and 16  weeks post - treatment and submersed in 10  % neutral buffered formalin .
tissues were embedded in paraffin , sectioned and placed on slides ( standard technique ) .
general morphological assessment was performed by staining the tissue sections with hematoxylin and eosin stains .
separate paraffin nerve sections from the same specimens were also subsequently stained for immunological assessment for neurofilament ( nf ) and s-100 expression .
ab34686 ) was followed with goat anti - rabbit alexa fluor 586 ( invitrogen ) as the secondary antibody .
next , mouse monoclonal nf - m ( santa cruz biotechnologies , clone : 3h11 ) was applied and followed with goat anti - mouse alexa fluor 488 as the secondary antibody .
one percent bsa in 1 pbs with 0.05  % tween-20 was used as the staining buffer .
axon density was manually counted for nf positive stained axons in seven imaging fields per time point .
measurement of toe - spread ability was used to quantify the weakening of the treated hind limb as compared to the contralateral control ( fig .  2 ) .
all animals demonstrated lack of toe spread with a score of 2 starting at 1  day post - treatment .
strong weakening of the toe - spread function continued for 14  days until gradual return to normal toe function by about 53  days post - treatment .
significant difference between the treated and control legs was seen for 28  days ( p  <  0.05 , calculated as compared to the contralateral untreated control . ) .
untreated control legs demonstrated a score of 0 throughout the post - treatment assessments ( not shown).fig . 
2toe - spread score of animals receiving fct at the tibial nerve ( n  =  10 ) .
when no error bars are shown , it indicates that the standard deviation is zero toe - spread score of animals receiving fct at the tibial nerve ( n  =  10 ) .
when no error bars are shown , it indicates that the standard deviation is zero the motor function ( walking ability ) assay measured the loss of function of the hind limb after cryotreatment .
motor function weakening was demonstrated with an average score of 2.7    0.3 in the treated leg starting at 1  day post - treatment ( fig . 
gradual return to normal walking ability was seen by 36  days post - treatment .
significant difference between the treated and control legs was noted for 18  days ( p  <  0.05 , calculated as compared to the contralateral untreated control ) . no abnormal or dysfunctional walking ability was observed after full recovery in hind limb function .
untreated control legs demonstrated a score of 0 throughout the post - treatment assessments ( not shown).fig . 
3motor function assay from animals that received treatment at the tibial nerve ( n  =  10 ) .
when no error bars are shown , it indicates that the standard deviation is zero motor function assay from animals that received treatment at the tibial nerve ( n  =  10 ) .
when no error bars are shown , it indicates that the standard deviation is zero histological assessments were performed on the treated nerves at 2 , 8 and 16  weeks post - treatment ( fig .  4 ) .
vacuolations were observed throughout the cross section ( green arrows ) , indicating degeneration and loss of axons .
condensation of the myelin sheath was observed , while the basal laminae remained intact at this time point .
4cross sections of h&e - stained nerves were assessed at b 2 , c 8 and d 16  weeks post - treatment .
blue arrow indicates regenerated axon cross sections of h&e - stained nerves were assessed at b 2 , c 8 and d 16  weeks post - treatment .
blue arrow indicates regenerated axon at 8  weeks post - treatment , significant axonal regeneration with concurrent elimination of macrophage infiltration was observed .
the newly regenerated axons appeared re - myelinated , while few degenerative axon fibers were noted .
few vacuolated areas were persisting at this time point . at 16  weeks post - treatment
no observable occurrences of neuromas and/or fibrosis were observed in any of the samples . staining for nf
( axons ) and s100 ( schwann cells ) showed analogous patterns of degeneration and regeneration as the h&e histology ( fig . 
nf ( green ) , s100 ( red ) and dapi ( blue ) were used to stain for axons , schwann cells and nuclei ( respectively ) . at 2  weeks , significant loss of nf - positively stained axons were observed .
5immunofluorescently stained cross section of a untreated control , b 2  weeks , c 4  weeks and d 8  weeks post - treated nerves .
green stain indicates positive for neurofilament , red stain indicates positive for s100 , and blue stain indicates nuclei immunofluorescently stained cross section of a untreated control , b 2  weeks , c 4  weeks and d 8  weeks post - treated nerves .
green stain indicates positive for neurofilament , red stain indicates positive for s100 , and blue stain indicates nuclei at 8  weeks post - treatment , early regeneration of axons was noticeable by the presence of positively stained axons for nf .
early regeneration of axons was observed by the presence of a small size and low - density population of stained cells .
few punctate staining for s100 was present , but there was more diffuse staining throughout the section . at 16  weeks
, complete regeneration of the treated nerve was noted with nf positively stained axons delimited by the s100 positively stained schwann cell
. further maturation of the axons was indicated by the increase in size of the axon diameter from the 8-week post - treated samples .
a count of axon density showed a significant reduction 2  weeks post - treatment followed by a gradual increase back to pre - treatment densities ( fig . 
axon density was measured as follows : untreated , 7,381    439 axons / mm ; 2  weeks post - treatment , 1,872    176 axons / mm ; 8  weeks post - treatment , 4,836    703 axons / mm ; and 16  weeks post - treatment , 6,501    531 axons / mm .
axon density at 2 and 8  weeks post - treatment showed significant ( p  <  0.05 ) difference from control , while at 16  weeks post - treatment it showed no difference ( p  >  0.05 ) compared to untreated control ( fig . 
6axon densities were compared in untreated and 2 , 8 and 16  weeks post - cryotreatment . * p  <  0.05 as compared to untreated control .
counts were measured in axons / mm 
  axon densities were compared in untreated and 2 , 8 and 16  weeks post - cryotreatment . * p  <  0.05 as compared to untreated control .
the present study demonstrated that the direct application of freezing temperature ( 56  c ) on a motor nerve will result in a temporary interruption of the target muscle function .
wallerian degeneration was indicated by the loss of axons observed in both h&e and immunofluorescence loss of axons and reduced staining of s100 .
this demonstrated equivalent histological results as seen with historical studies using various freezing temperatures ( kerns et al .
histological analyses suggested that the axons and schwann cells became disrupted , while the injury left the epineurium and perineurium intact .
the schwann cells are able to form the bands of bngner along the endoneurial tube , which aid in guiding regenerating axons to their target tissue .
the temporary loss of axons was reflected in the functional loss of the treated hind limb .
disruption of the hind limb nerve conduction resulted in a deficit in toe - spread function and walking gait ( see figs .  2 , 3 ) .
toe - spread data showed increased weakening and longer duration compared to the walking motor function data .
this difference arises from the different muscle groups being measured in each of these assays .
toe function relies on the function of the more distal muscle groups of the hind limb ( i.e. , extensor digitorum longus and digiti minimi abductor ) , while the motor function relies on both the distal and proximal muscle groups of the hind limb .
this difference in motor function and toe - spread data reflects the focused nature of the treatment to the targeted nerve .
full axonal regeneration was confirmed with complete restoration of axon density and morphological recovery comparable to the untreated nerve ( see fig . 
complete physiological function did not necessitate complete axonal regeneration . at 8  weeks , normal function was observed coincident with a 34  % decrease in level of normal axonal density ( see fig . 
there is sufficient axonal signaling to the target muscle groups to restore normal movement without complete axonal regeneration .
the physiological assays currently used in this study did not capture the strength of the treated hind limb .
a more elaborate study should further reveal the correlation between axon density , muscle tetanic forces and physiological function .
this proposed method of cooling a motor nerve to induce a temporary injury involving second degree wallerian degeneration and reinnervation to reduce muscle contraction can be used in the clinical setting .
in particular , this technology can be used to reduce dynamic facial wrinkles with fct to specific facial motor nerve branches .
further use of this platform technology can expand to other indications , including movement disorders which involve hyperactive muscle functions such as dystonia , spasms and general weakening of a muscle control .
some of the current treatments for these types of movement disorders involve the use of neurotoxins and drugs which inhibit the nerve signaling to the muscle , sometimes with unwanted side effects .
fct treatment of the motor nerve can potentially be used as a drug - free method of relaxing the specific target muscle .
reduction of muscle function with the use of fct is shown to be temporary followed by a normal return of function .
 application of freezing temperatures to the temporal branch of the facial nerve can temporarily inhibit motor nerve conduction , resulting in inhibition of voluntary contraction of the frontalis and glabella muscle groups . 
 this feasibility study demonstrates the reduction in motility of muscle groups through application of low temperatures to nerves in a rat model . 
 twenty - seven adult female sprague  
 dawley rats received cryotreatment to the tibial nerve of the hind limb , and the contralateral limb was left untreated as a negative control . 
 the use of a cold temperature application ( 59    8  c for 60  s ) onto the rat tibial nerve resulted in temporary reduction of physiological function of the hind limb . 
 histological observations of the nerve revealed demyelination and axonal degeneration by 2  weeks post - treatment followed by complete axonal regeneration and remyelination at 16  weeks . 
 application of low temperatures to peripheral motor nerves resulted in temporary denervation and loss of function of the treated hind limb . 
 low temperature treatment on motor nerves did not result in any permanent or long - term changes to function and structure of the nerves . 
Introduction
Methods and materials
Results
Discussions/conclusions