tbe ( 2,2,2- tribomoethanol 97% , c2h3br3o , mw 282.8 ) , tert - amyl alcohol ( 99% ) and methyl orange were purchased from sigma aldrich , ( st .
hp--cd ( kleptose hpb , mw 1440 ) was obtained from a gift from roquette lestrem , france .
combititrant 5 and methanol ( 99.8% ) were purchased from merck ( auckland , new zealand ) .
atropine was obtained as phoenix atropine injection from provet nz ltd ( christchurch , new zealand ) .
hematoxylin and eosin dyes for histological examination were obtained from surgipath medical ( illinois , usa ) .
tbe is reported to have a solubility of 1 in 40 parts water at 40c , soluble in alcohol , ether , bezene and chloroform and very soluble in tert - amyl alcohol and is supplied as a white crystalline powder .
a 1.25% w / v tbe anesthetic solution was produced following the method recommended by the animal welfare office , university of otago , by dissolving 0.5 g tbe in 0.25 g tert - amyl alcohol then diluting to 40 ml with 0.9% nacl .
dilution of the tert - amyl alcohol solution produced a precipitate , which redissolved with stirring for 30 - 60 min on a magnetic stirrer .
the final solution was filtered ( 0.2 m ) into an amber glass bottle , protected from light by covering the vial with foil and stored at 4c .
the water content of hp--cd samples ( 0.05 g , n = 5 ) was determined by karl fischer titration ( 736 gp titrino , metrohm , usa ) .
the mixture was calibrated with 10 l of distilled water until a consistent value was obtained .
the water content of the hp--cd solid was calculated to be 8.4% w / w .
tbe has no appreciable ultra violet absorbance and so direct measurement of the binding constant for tbe and hp--cd could not be performed .
an approximate phase solubility diagram was constructed by addition of small aliquots ( 5 - 30 mg ) of tbe to hp--cd solutions ( 0 - 0.035 m ) until no further tbe would dissolve .
solutions were protected from light to reduce the risk of chemical degradation during the observation period and stored at 20c .
temperature was monitored using a hobo data logger ( onset computer corp . , bourne , ma , usa ) .
cyclodextrin formulations were prepared aseptically no more than 2 days prior to being used in the animal study .
the cyclodextrin tbe formulation was prepared by dissolving 0.25 g tbe with 0.344 g hp--cd ( weight corrected for water content ) in 20 ml to give a concentration of 1.25% w / v in 0.012 m hp--cd .
this mixture was dispersed using a magnetic stirrer ( ~1 h ) and once fully dissolved , the solution was filtered ( 0.2 m ) and stored at 4c and protected from light until administered .
the procedures described had prior approval from the university of otago animal ethics committee , dunedin and conformed to the university of otago code of ethical conduct for the manipulation of animals ( 1987 ) and the animal welfare act ( 1999 ) in new zealand .
specific pathogen free , adult , female balb / c mice of the same approximate weight ( 19 - 21 g ) were maintained in group housing and acclimatized to the environment for at least 7 days prior to being used in the study .
animals were offered food and water ad libitum , and both were not withheld prior to , or following , anesthesia .
animals were removed from group housing , weighed and placed in individual cages on heating pads to maintain body temperature .
atropine ( 0.05 mg / kg ) was administered subcutaneously 30 min prior to administration of the anesthetic formulations . the cyclodextrin tbe formulation and the standard tbe solution
were compared using the dose regimen of 260 mg / kg tbe administered through the ip route ( n = 6/group ) .
parameters observed in the animals were depth of anesthesia , time to righting reflex loss and gain . the anesthetic efficacy of each formulation was assessed in the mice by monitoring several reflex responses . the time after administration of the anesthetic
to loss of righting reflex , the animal 's reflex to roll onto its front when placed on its back , was recorded .
the intensity of the tail pinch reflex was assessed after pinching the tail of the mouse with plastic forceps .
the intensity of the pedal withdrawal reflex was assessed when the interdigital skin on the fore and hind feet were pinched with plastic forceps .
tail and pedal withdrawal responses were scored on a scale from 0 to 3 ( 0 = no response through to 3 = strong response as in a conscious animal ) . the reflex scores for each time point
were summed to give a combined reflex score ( crs ) of 0 - 9 ( 0 = surgical anesthesia , 9 = fully responsive ) for each animal .
14 days following administration of the anesthetic formulations , a necropsy was performed and tissue samples of the liver and gut were collected for histological examination .
the tissue samples were fixed with 10% formalin , embedded in paraffin and sectioned with a leica rm2025 rotary microtome ( nussloch , germany ) to 3 m thick sections .
statistical analysis was performed using statistical package for the social sciences software ( ibm , version 19 ) .
the comparison between respiratory rate with standard and cyclodextrin tbe formulations was undertaken by repeated measures analysis of variance model .
the time to loss and gain , of righting reflex was compared between the two treatment groups using an independent samples t - test .
tbe ( 2,2,2- tribomoethanol 97% , c2h3br3o , mw 282.8 ) , tert - amyl alcohol ( 99% ) and methyl orange were purchased from sigma aldrich , ( st .
hp--cd ( kleptose hpb , mw 1440 ) was obtained from a gift from roquette lestrem , france .
combititrant 5 and methanol ( 99.8% ) were purchased from merck ( auckland , new zealand ) .
atropine was obtained as phoenix atropine injection from provet nz ltd ( christchurch , new zealand ) .
hematoxylin and eosin dyes for histological examination were obtained from surgipath medical ( illinois , usa ) .
tbe is reported to have a solubility of 1 in 40 parts water at 40c , soluble in alcohol , ether , bezene and chloroform and very soluble in tert - amyl alcohol and is supplied as a white crystalline powder .
a 1.25% w / v tbe anesthetic solution was produced following the method recommended by the animal welfare office , university of otago , by dissolving 0.5 g tbe in 0.25 g tert - amyl alcohol then diluting to 40 ml with 0.9% nacl .
dilution of the tert - amyl alcohol solution produced a precipitate , which redissolved with stirring for 30 - 60 min on a magnetic stirrer .
the final solution was filtered ( 0.2 m ) into an amber glass bottle , protected from light by covering the vial with foil and stored at 4c .
the water content of hp--cd samples ( 0.05 g , n = 5 ) was determined by karl fischer titration ( 736 gp titrino , metrohm , usa ) .
the mixture was calibrated with 10 l of distilled water until a consistent value was obtained .
the water content of the hp--cd solid was calculated to be 8.4% w / w .
tbe has no appreciable ultra violet absorbance and so direct measurement of the binding constant for tbe and hp--cd could not be performed .
an approximate phase solubility diagram was constructed by addition of small aliquots ( 5 - 30 mg ) of tbe to hp--cd solutions ( 0 - 0.035 m ) until no further tbe would dissolve .
solutions were protected from light to reduce the risk of chemical degradation during the observation period and stored at 20c .
temperature was monitored using a hobo data logger ( onset computer corp . , bourne , ma , usa ) .
cyclodextrin formulations were prepared aseptically no more than 2 days prior to being used in the animal study .
the cyclodextrin tbe formulation was prepared by dissolving 0.25 g tbe with 0.344 g hp--cd ( weight corrected for water content ) in 20 ml to give a concentration of 1.25% w / v in 0.012 m hp--cd .
this mixture was dispersed using a magnetic stirrer ( ~1 h ) and once fully dissolved , the solution was filtered ( 0.2 m ) and stored at 4c and protected from light until administered .
the procedures described had prior approval from the university of otago animal ethics committee , dunedin and conformed to the university of otago code of ethical conduct for the manipulation of animals ( 1987 ) and the animal welfare act ( 1999 ) in new zealand .
specific pathogen free , adult , female balb / c mice of the same approximate weight ( 19 - 21 g ) were maintained in group housing and acclimatized to the environment for at least 7 days prior to being used in the study .
animals were offered food and water ad libitum , and both were not withheld prior to , or following , anesthesia .
animals were removed from group housing , weighed and placed in individual cages on heating pads to maintain body temperature .
atropine ( 0.05 mg / kg ) was administered subcutaneously 30 min prior to administration of the anesthetic formulations . the cyclodextrin tbe formulation and the standard tbe solution
were compared using the dose regimen of 260 mg / kg tbe administered through the ip route ( n = 6/group ) .
parameters observed in the animals were depth of anesthesia , time to righting reflex loss and gain .
the anesthetic efficacy of each formulation was assessed in the mice by monitoring several reflex responses .
the time after administration of the anesthetic to loss of righting reflex , the animal 's reflex to roll onto its front when placed on its back , was recorded .
the intensity of the tail pinch reflex was assessed after pinching the tail of the mouse with plastic forceps .
the intensity of the pedal withdrawal reflex was assessed when the interdigital skin on the fore and hind feet were pinched with plastic forceps .
tail and pedal withdrawal responses were scored on a scale from 0 to 3 ( 0 = no response through to 3 = strong response as in a conscious animal ) .
the reflex scores for each time point were summed to give a combined reflex score ( crs ) of 0 - 9 ( 0 = surgical anesthesia , 9 = fully responsive ) for each animal .
14 days following administration of the anesthetic formulations , a necropsy was performed and tissue samples of the liver and gut were collected for histological examination .
the tissue samples were fixed with 10% formalin , embedded in paraffin and sectioned with a leica rm2025 rotary microtome ( nussloch , germany ) to 3 m thick sections .
statistical analysis was performed using statistical package for the social sciences software ( ibm , version 19 ) .
the comparison between respiratory rate with standard and cyclodextrin tbe formulations was undertaken by repeated measures analysis of variance model .
the time to loss and gain , of righting reflex was compared between the two treatment groups using an independent samples t - test .
the solubility of tbe in water is reported to be 1 in 40 ( 2.5% w / v ) at 40c . in the present study
, we observed the solubility of tbe to increase linearly with increasing hp--cd concentration [ figure 1 ] .
the solubility in the absence of hp--cd was 28 mg / ml ( 2.8% w / v ) and temperature recorded over the study period was 21  1.5c .
the al shaped diagram with a slope of greater than 1 suggested a higher order interaction with respect to the drug ( > 1:1 ) exists between tbe and hp--cd . since tbe is a small molecule it is possible that 2:1 or greater molar ratio tbe : hp--cd complex was formed . using equation 1 ,
least squares linear regression produced a slope = 1.608 ( standard error = 0.065 ) ; intercept = 0.1037 ( se = 0.0037 ) ; r = 0.993 where s0 = solubility ( m ) of tbe in the absence of hp--cd and the slope is from the tbe solubility versus hp--cd concentration graph [ figure 1 ] .
a notable difference between the preparations of the formulations was that tbe could be readily dissolved directly in the cyclodextrin solution prior to use , whereas for the standard formulation tbe required dissolution in an organic solvent followed by dilution with normal saline .
when the organic solution was diluted , tbe precipitation was observed and time was required to allow this to re - dissolve prior to use .
the use of cyclodextrin formulations can reduce precipitation in vivo as discussed by brewster and loftsson .
the relationship between the solubility of a drug that forms a complexation with a cyclodextrin is commonly linear .
consequently , upon administration , dilution of the drug - cyclodextrin complex causing dissociation should also occur in a linear fashion , and without precipitation .
this is in contrast to preparations made using organic solvents where the solubilization of the drug is based on a non - linear relationship and so upon dilution following in vivo administration , the ability of organic solvents to solubilize the drug is rapidly reduced leading to precipitation . owing to the disparity in doses reported in the literature ( 125 - 500 mg / kg )
, a preliminary study was undertaken to determine an appropriate dose of tbe to achieve anesthesia in mice .
consistent with other studies , at a dose of 260 mg / kg there was good surgical anesthesia , however there was also some evidence of respiratory depression ( i.e. vocalizations , labored breathing ) .
consequently , atropine was administered 30 min prior to anesthesia ( 0.05 mg / kg ) by subcutaneous injection .
a comparison of anesthesia between the standard tbe solution and the cyclodextrin tbe formulation figure 2 shows the trend for the standard formulation to produce a deeper level of anesthesia , although the difference was not statistically significant .
the lowest crs attained was 0 for the standard formulation and 2 for the cyclodextrin formulation [ figure 2 ] .
short and variable anesthetic times with tbe ( dose 300 mg / kg ) have been reported by avila et al . and
these authors also comment that the duration of anesthesia was sometimes too short to conduct their intended measurements .
the effectiveness of the standard tbe formulation compared to the cyclodextrin formulation may be attributed to the presence of organic solvent ( concentration = 0.625% in the injected solution ) , which is known to cause central nervous system depression and ataxia on its own , and which is not present in the cyclodextrin tbe formulation .
complexation of the tbe with the cyclodextrin would also result in reduced free drug concentrations compared to the standard formulation .
it may be that rather than a comparison of the same dose ( 260 mg / kg ) between the two formulations , a higher dose of the cyclodextrin formulation may be required to have the same effect as the standard formulation .
binding of tbe by hp--cd supports the observation of reduced pharmacodynamic effect in the present study .
however , stella and rajewski reported that release of drugs from cyclodextrin complexes occurs very rapidly once administered to the body .
recovery from the tbe anesthetic was more rapid for the cyclodextrin group as all mice had regained a crs of 9 by 30 min following administration compared to up to 1 h for the standard tbe formulation .
the time to loss and gain of righting reflex reflects the level of anesthesia reached with the two formulations .
results from observation of the righting reflex [ table 1 ] show that this reflex is lost approximately 30 s faster with the standard formulation ( p = 0.006 ) and it appears to take longer to return , although this result was not significant . at a relatively high tbe dose of 400 mg / kg ,
the time interval between loss and regain of righting reflex has reported to be between 0 min and 120 min , with a mean of 21.5 min .
influence of anesthetic formulation on combined reflex score for mice treated with ( a ) standard tribromoethanol or ( b ) hydroxypropyl--cyclodextrin tribromoethanol ( n = 6/group ) . dose of tribromoethanol was 260 mg / kg + atropine 0.05 mg / kg .
crs score 0 = surgical anesthesia and 9 = fully responsive comparison of times to loss and gain of righting reflex between the standard tribromoethanol formulation and hydroxypropyl--cyclodextrin tribromoethanol formulation variable anesthetic effect is reported in the literature for tbe and comparisons may be confounded by the variety of doses used , animal characteristics ( age , sex and species ) and method of assessment of anesthesia .
for example , a gender difference is reported for tbe with female mice having longer durations of anesthesia .
the current experiments were performed to reduce sources of variability by using an inbred strain of mice , one gender ( females ) of similar age and weight and performing the experiments at the same time of day to reduce variation due to circadian rhythm .
it is also noted in the present study that there was considerable individual variation in response to the anesthetic , with the group of mice that received the cyclodextrin formulation showing less variation [ figure 2 ] .
the observed differences in response following the injection of tbe could be in part due to the variable placement of the ip injection within the peritoneum .
injection of the formulations adjacent to gut vasculature may lead to a more rapid and higher absorption , as diffusional distances for tbe are less .
such variability in absorption of substances from the peritoneal cavity is known to vary between subjects , as has been seen with the peritoneal absorption of insulin in humans .
there has also been criticism of the use of the ip route as it is easy to cause damage to the intestine and visceral organs during administration of the injection .
the use of a cyclodextrin formulation instead of one that includes an organic solvent may make it feasible for administration to be performed subcutaneously rather than the ip route ; however the suitability of the subcutaneous site for tbe remains to be investigated .
some researchers report high rates of post - anesthesia mortality , peritonitis , intestinal ileus and abdominal adhesions , whereas others have used tbe extensively with few adverse effects .
the mice in the current study were monitored daily for 14 days after administration of the anesthetic for clinical signs of illness , i.e. weight loss , reduction in water intake , abnormal behavior . at day 14
no evidence of inflammation or gross lesions was identified in the abdominal cavity of the animals .
tissue samples were taken from the spleen , kidney , bowel and liver for histological examination .
there was no evidence of inflammation or fibrosis found in any of the tissues examined and there was no difference between the two tbe formulations investigated [ figure 3 ] .
norris and turner investigated the use of tbe in mongolian gerbils at varying concentrations ( 1.25 - 2.25% ) and doses ( 225 - 450 mg / kg ) and found that mortality rate increased with the use of the 2.25% solution and at higher doses for the 1.25% solution .
the use of the lower concentration ( 1.25% ) solution and the relatively low dose rate in the present study may be a contributing factor why no inflammation was observed in the tissues .
in addition , the absence of pathological findings may be because of the period of time between the animals receiving the formulation and the euthanasia .
the post mortem conducted 14 days after anesthetic administration may have provided time for any inflammation to resolved , however thompson et al
. investigated histopathological changes to the liver , spleen , heart and kidney 3 and 6 h after the onset of anesthesia following an ip injection of tbe in mice ( dose 160 mg / kg ) and also report no detectable pathologic changes in the tissue .
photomicrographs of bowel section from mice administered ( a ) standard tribromoethanol formulation and ( b ) cyclodextrin tribromoethanol formulation .
sections of liver parenchyma 14 days after administration of ( c ) standard tribromoethanol formulation and ( d ) cyclodextrin tribromoethanol formulation .
v = vein , hepatocyte nuclei are stained a blue / black colour ( arrow ) and red blood cells visible in the sinusoids
tbe could be formulated as a 1.25% w / v solution 0.035 m hp--cd in a single step without the need for an organic solvent .
this study has also identified the value in using atropine in addition to tbe for anesthesia .
the reduced anesthetic effect of the cyclodextrin formulation compared to the standard formulation may have been due to tbe complexation with the cyclodextrin complex .
no evidence of peritonitis or abdominal adhesions were observed with either formulation , suggesting that freshly prepared tbe solutions ( standard or hp--cd ) at a concentration of 1.25% were not significantly irritant .
 objective : efficacy and safety concerns have been raised in the literature with the use of tribromoethanol ( tbe ) ( avertin ) for anesthesia in rats and mice when administered by intraperitoneal ( ip ) injection . despite the controversy , it remains in common usage as an anesthetic agent in laboratory rodents for short - term surgical procedures . 
 cyclodextrins have been shown to improve drug solubility and were investigated here as an improved anesthetic formulation for mice.materials and methods : the phase solubility of tbe with hydroxypropyl--cyclodextrin ( hp--cd ) was estimated . 
 the efficacy of two anesthetic regimens was compared in this study ; the conventional tbe formulation solubilized in tert - amyl alcohol and a hp--cd formulation containing tbe . 
 mice ( n = 6 ) were administered the formulations by ip injection and the pharmacodynamic parameters of time to induction of anesthesia , duration of anesthesia and recovery time were measured using a combined reflex score ( crs).results and discussion : phase solubility studies showed a linear increase in the solubility of tbe with increasing hp--cd concentration and suggested > 1:1 binding of the drug in the cyclodextrin complex . at a dose of 260 mg 
 / kg the standard tbe formulation appeared to produce deeper anesthesia than the cyclodextrin formulation , with a minimum average crs of 1.8 compared with 5.2 . 
 no post - mortem pathology was observed in mice that received either the conventional or cyclodextrin formulation.conclusion:the cyclodextrin tbe formulation did not conclusively provide an improved anesthetic response at a dose of 260 mg / kg compared with the conventional formulation . 
 the improved solubility of tbe with hp--cd and the reduced variability in anesthetic response warrants the further investigation of this formulation . 
 this study has also identified the value of using the anticholinergic atropine in association with tbe for anesthesia . 
Materials and Methods
Chemicals
TBE anesthetic solution
Cyclodextrin formulation
Administration of formulations and assessment of anaesthesia
Statistical analysis
Results and Discussion
Conclusion