Development of a simple method for differential delivery of volatile anesthetics to the spinal cord of the rabbit.

Emulsified volatile anesthetic can be directly injected into the circulation and eliminated from blood through lungs. Taking advantage of the unique pharmacokinetics of the emulsified volatile anesthetics, we aimed to develop a less traumatic method to differentially deliver them to the spinal cord of rabbit. Sixteen New Zealand White rabbits were randomly assigned to the isoflurane or sevoflurane group. A catheter was placed into the descending aorta, and emulsified isoflurane (8mg/kg/h) or sevoflurane (12mg/kg/h) was given respectively. The concentration and partial pressure of the anesthetics in the jugular and femoral vein were measured. Our results showed that the partial pressure for isoflurane was 3.91±1.11 mmHg and 12.61±1.60 mmHg (1.0MAC), and for sevoflurane was 3.89±1.00 mmHg and 19.92±1.84mmHg (1.0MAC), in the jugular vein and femoral vein, respectively. There was significant difference between jugular and femoral vein partial pressure for both isoflurane and sevoflurane groups (both P < 0.001). In conclusion, a simple and minimally invasive method has been successfully developed to selectively deliver isoflurane and sevoflurane to the spinal cord in the rabbit. Before the anesthetics taking action on the brain, 69% of isoflurane and 81% of sevoflurane were removed through lungs. This method can be used to investigate sites and mechanisms of volatile anesthetic action.

Introduction

Volatile anesthetics can induce a variety of reversible, clinically important effects such as amnesia, hypnosis and immobility [1, 2]. To differentiate whether an anesthetic effect comes from brain, spinal cord or both, methods that selectively deliver the anesthetic to target tissues are needed [3, 4]. Previous research has selectively delivered anesthetics to the brain, upper or lower torso of goat, dog and rabbit using cardiopulmonary bypass technology [5-7]. However, our model permits selective perfusion of the spinal cord versus brain that is less costly and labor intensive while obviating most of the trauma associated with prior models.

Rabbits have a unique spinal cord circulation in that each spinal cord segment is supplied by a single corresponding radicular artery arising from the aorta [8, 9]. In addition, the blood supply to the thoracolumbar region (below T3) of spinal cord originates from the thoracic and abdominal aorta in the rabbit [10-12]. Volatile anesthetics can be dissolved in an emulsifiying agent, which produces anesthesia as effectively as a vaporized and inhaled one [13, 14]. Similar to intravenous drugs, emulsified volatile anesthetics can be directly injected into the circulation, but eliminated through lungs [15, 16]. In this report, we aimed to establish a simpler, less labor intensive, and less traumatic method that can be used to selectively deliver volatile anesthetics (isoflurane and sevoflurane) to the spinal cord in rabbit.

Materials and methods

Emulsified isoflurane and sevoflurane preparation

Isoflurane and sevoflurane emulsion were prepared according to a previously described formula [17, 18]. In brief, 1.6mL of liquid isoflurane or sevoflurane (Abbott, Shanghai, China) and 18.4mL of 30% intralipid (Baxter, Suzhou, China) were injected into a sealed sterile vial. Then the vial was violently shaken for 10min by a vibrator to solubilize isoflurane or sevoflurane into the intralipid. The 8% emulsified isoflurane and sevoflurane were stored in 4°C refrigerator before use.

Animal preparation and surgical procedures

The study protocol was approved by the Institutional Animal Care and Use Committee of Sichuan Provincial People's Hospital. Sixteen New Zealand White rabbits (male and female) weighing 2.0–3.0kg were randomly assigned to the isoflurane or sevoflurane group (8 each). After intravenous injection of 30 mg/kg pentobarbital sodium into the left marginal ear vein, an ID 3.0# cuffed endotracheal tube was inserted. Before intubation, 1% dicaine gel was coated to the endotracheal tube for airway topical anesthesia, which could increase the tolerance for endotracheal tube [18, 19]. All rabbits were mechanically ventilated with 95% O2 using an animal ventilator (Chengdu Techman Software Co.Ltd, Chengdu, China), with the tidal volume 8ml/kg, 40 breaths/min, to maintain the arterial carbon dioxide pressure (PaCO2) between 35 to 45 mmHg. Local anesthetic (1% lidocaine+0.5% ropivacaine) were injected into the skin before surgical exposure. A 22-gauge IV catheter (BD company, Sandy Utach, USA) was individually inserted into the left central ear and femoral arteries respectively to monitor the blood pressure in the upper and lower torso of the rabbit.

An epidural catheter (TuoRen Medical Instrument Co., Ltd, Xinxiang, China) was placed into the descending aorta from the right femoral artery (Fig 1). It was used to deliver emulsified isoflurane or sevoflurane to the spinal cord. The change of resistance was monitored during the catheter passing cranially along the aorta. The catheter would be withdrawn 0.5–1.0 cm, when the increased resistance was recorded. The placement of catheter tip was confirmed by autopsy after the experiment. In our study, the rabbit would be excluded from data analysis if the tip was at the aortic arch level. The length of catheter between catheter tip and inguinal fold was measured.

Fig 1

Diagram of the differential anesthetic delivery rabbit model.

The infusion rate was 8mg/kg/h in the isoflurane group and 12mg/kg/h in the sevoflurane group by a microinfusion pump. When the end-tidal isoflurane concentration (ETISO) or end-tidal sevoflurane concentration (ETSEVO) was stable and maintained for 20min, emulsified volatile anesthetic was considered to reach steady state condition [15]. There were two methods to avoid re-breaching of isoflurane or sevoflurane into the brain: (a) a higher inspiratory flow 2L/min (normal minute ventilation (MV) of rabbit is about 1L/min) (b) an anesthetic absorber made of activated charcoal in the inspiratory limb of the circuit.

Lactated Ringer’s solution (6ml/kg/h) was administered through the left marginal ear vein in all rabbits. Rectal temperature was monitored and maintained at 37.0±1.0°C. During the experiment, heart rate (HR), mean arterial pressure (MAP), electrocardiograph (ECG) and pulse oxygen saturation (SpO2) were measured with the BL-420E+ Data Acquisition and Analysis System (Chengdu Techman Software Co.Ltd, Chengdu, China). The ETISO, ETSEVO and ETCO2 (end-tidal CO2) were monitored with an anesthetic gas analyzer M1026B (Philips Medizin Systeme Boblingen GmbH, Boblingen, Germany). Isoflurane or sevoflurane 1.0 MAC was permitted during the experiment, and was discontinued to wash out volatile anesthetics, 15-20min before the infusion of the emulsified volatile anesthetics.

Collection of blood samples and gas chromatograph

Once the end-tidal concentration of isoflurane or sevoflurane was stable and maintained for 20min, 5-6ml blood was rapidly withdrawn from the jugular and femoral vein to determine the isoflurane or sevoflurane blood concentration (C). Samples were analyzed using a gas chromatograph Agilent 4890D (Tegent Technology Ltd., Hong Kong, China) with two-stage headspace equilibration methods [15]. The partial pressure of isoflurane and sevoflurane (P) in different blood samples were calculated by the equation of P = (C/λb/g×760 mmHg) (λb/g = blood/gas partition coefficient). The C and P of all samples were measured by a technician who was blinded to the samples. In our previous study, the partial pressure of 1.0 MAC (minimum alveolar concentration) isoflurane was 11.66±1.10 mmHg and the partial pressure of 1.0 MAC sevoflurane was 18.86±1.12 mmHg.

Statistical analysis

Statistical analyses were performed using SPSS 22.0. Each continuous variable was analyzed for its normal distribution and reported as mean ± SD. Repeated measures ANOVA was used to evaluate MAP and HR among different time points in two groups. Other continuous variable data (the weight of the rabbit, the length of the catheter and the partial pressure of isoflurane and sevoflurane) were analyzed by student’s t test. χ2 test was used to evaluate the position of catheter tips. Pearson correlation coefficient was used to measure the linear relationship between λb/g and the volume (mL) of the emulsified isoflurane or sevoflurane consumed. A P value of less than 0.05 was considered statistically significant. A Bonferroni correction was made when necessary to correct for multiple testing.

Results

Full data sets were collected from sixteen rabbits. The mean weight of the rabbits was 2.38±0.26 kg in the isoflurane group and 2.46±0.24 kg in the sevoflurane group, which were not significantly different (P = 0.532). The mean lengths of the inserted catheter (between catheter tip and inguinal fold) were 25.13±1.07 cm in the isoflurane group and 25.36±0.86 cm in the sevoflurane group. In the isoflurane group, two catheters (25%) were located at T2 and six (75%) at T3. In the sevoflurane group, four catheters (50%) were placed at T2 and four (50%) at T3 (Table 1). There was no significant difference with regard to the length of the catheter (P = 0.608) and the position of the catheter tips (P = 0.631) between two groups.

Table 1

The position of catheter tips in two groups.

Group	T2	T3	Total
Isoflurane	2 (25%)	6 (75%)	8
Sevoflurane	4 (50%)	4 (50%)	8
Total	6	10	16
P value	0.608

No significant difference was found regarding MAP of the central ear artery among three different time points in two groups (P = 0.169 for isoflurane group; P = 0.390 for sevoflurane group). But MAP of the femoral artery at the midpoint of the infusion was significantly reduced compared with the other two time points (after induction and at the end of the study) in two groups (both P<0.001). The trend of MAP change between central ear artery and femoral artery was significantly different during the whole experiment in both groups (P<0.001). However, there was no significant difference in the MAP between central ear artery and femoral artery during the whole experiment in two groups (P = 0.062 for isoflurane group; P = 0.169 for sevoflurane group) (Table 2). No significant difference was found with regard to HR among three time points in the isoflurane group (P = 0.091) and sevoflurane group (P = 0.076) (Table 3).

Table 2

The mean atrial pressure (MAP, mmHg) of femoral and central ear artery among different time points in two groups (mean ± SD).

Group	Position	After induction	Midpoint of infusion	End of the study
Isoflurane	Central ear artery	90.4±2.3	86.3±5.5	87.5±4.5
	Femoral artery	92.3±3.6	74.6±6.1#&	88.4±4.4
Sevoflurane	Central ear artery	88.1±5.1	84.6±7.5	84.3±5.4
	Femoral artery	90.3±5.0	71.4±4.3#&	86.1±4.8

#P<0.001, midpoint of infusion vs after induction

&P<0.001, midpoint of infusion vs end of the study

Table 3

The heart rate (HR, beats/min) among different time points in two groups (mean ± SD).

Group	After induction	Midpoint of infusion	End of the study
Isoflurane	203±11	201±5	212±11
Sevoflurane	215±11	207±9	220±13

The partial pressure of isoflurane was 3.91±1.11 mmHg in the jugular vein and 12.61±1.60 mmHg (1.0MAC) in the femoral vein. The partial pressure of sevoflurane was 3.89±1.00 mmHg in the jugular vein and 19.92±1.84mmHg (1.0MAC) in the femoral vein. The partial pressure in the jugular and femoral vein was significantly different for both the isoflurane and sevoflurane groups (both P<0.001). The ratio of partial pressure of isoflurane and sevoflurane between jugular vein and femoral vein is shown in the Table 4.

Table 4

The partial pressure of isoflurane and sevoflurane (mmHg) in the jugular (Pj) and femoral vein (Pf) in two groups (mean ± SD).

Group	Pj jugular vein	Pf femoral vein	Ratio1 (Pj/ Pf)%	Ratio2 (Pf/ Pj)	P value
Emulsified isoflurane (8mg/kg/h)	3.91±1.11	12.61±1.60	31.28±9.17%	3.48±1.13	<0.001
Emulsified sevoflurane (12mg/kg/h)	3.89±1.00	19.92±1.84	19.40±4.46%	5.34±1.18	<0.001

There was significant positive correlation between λb/g and volume of the emulsified anesthetics delivered (r = 0.935, P<0.001 for isoflurane group; r = 0.919, P = 0.001 for sevoflurane group). The Linear regression equation was y = 0.0869x+0.9688 (R2 = 0.8723) in the isoflurane group (Fig 2) and y = 0.0526x+0.3889 (R2 = 0.8452) in the sevoflurane group (Fig 3) (y = volume and x = λb/g).

Fig 2

The positive correlation between isoflurane blood/gas partition coefficient and the volume of emulsified isoflurane delivered.

Fig 3

The positive correlation between sevoflurane blood/gas partition coefficient and the volume of emulsified sevoflurane delivered.

Discussion

Rabbit, with its medium size and homosegmental blood supply to the spinal cord, is an ideal animal for selective spinal cord anesthetic delivery [10-12]. With the catheter inserted into the descending aorta, isoflurane and sevoflurane emulsion were able to be preferentially delivered to the thoracolumbar (below T3) region of the spinal cord. Before acting on the brain, the majority of anesthetic was eliminated through lungs. Using this model, 69% of isoflurane, or 81% of sevoflurane was eliminated by the lungs before reaching the brain. Therefore, the torso circulation below the T3 level was selectively perfused with anesthetic compared to the upper spinal cord and brain in our study.

When volatile anesthetic reached a steady state condition in the body, the partial pressure in the alveolar, blood and central nervous system is presumed to be in equilibrium with one another [15, 20]. In our study, to be specific, the partial pressure in the jugular and femoral veins reflect that in the brain and spinal cord, respectively, under a steady state condition. Our results showed that the isoflurane partial pressure in the femoral vein was about 3.5-fold of that in the jugular vein, and the ratio of sevoflurane was 5.3-fold. In other words, the partial pressure of isoflurane and sevoflurane in the spinal cord were 3.5-fold and 5.3-fold of that in the brain tissue respectively. Thus, our model could differentially deliver emulsified isoflurane and sevoflurane to the spinal cord in rabbit.

In Yang’s goat model [18], using similar technologies, emulsified isoflurane was selectively delivered to the spinal cord, with an approximate 46% reduction in the isoflurane partial pressure in the brain [18]. In our present study, brain concentrations of isoflurane and sevoflurane were only 31% and 19% of the spinal cord concentrations, respectively. Therefore, our rabbit model was more selective compared to the previous goat model using emulsified isoflurane. There were several possible reasons: (a) The oxygen consumption per kilogram of rabbit is larger than that of goat, and MV/kg of rabbit is about 2 times than that of goat [21]. It has established that increasing MV might contribute to remove volatile anesthetics from blood via lungs [22]; (b)Two methods mentioned (higher inspiratory flow and anesthetic absorber) were used to avoid re-breaching of isoflurane or sevoflurane; (c) In our study, sevoflurane was more rapidly eliminated from body than isoflurane, because of its lower blood gas solubility (λb/g of sevoflurane = 0.069 versus λb/g of isoflurane = 1.37) [23].

It is well known that the λb/g of 30% intralipid is remarkably larger than the λb/g of both isoflurane and sevoflurane. So λb/g of isoflurane and sevoflurane would significantly increase with infusion of lipid emulsion in our study. Our results indicated that infusion of 1 ml emulsified volatile anesthetics increased λb/g by 0.0869 for isoflurane and by 0.0526 for sevoflurane at 37°C. Some discrepancies in the literature showed that 1 ml of 8% emulsified isoflurane raised λb/g by 0.0176 in the goat [18] and 0.0139 in the dog [15]. Obviously, the blood volume of rabbit is significantly less than that of goat or dog [22]. Based on “volume fraction partition coefficient” theory, when 1ml of lipid emulsion was administrated to those three animals, the increase of rabbit λb/g of isoflurane might be larger than that of goat or dog [24]. However, there was no report published regarding λb/g of sevoflurane.

The limitations of our study are as follows: the volatile anesthetics (VAs) concentration in the brain can not be regulated, and the lower VAs partial pressure in the brain (0.2–0.3MAC) could keep the rabbits awake during the infusion period, which might bring animal welfare concerns. However, we believe there are three reasons to eliminate most of the animal welfare issues. (i) In our model, all rabbits were under isoflurane or sevoflurane general anesthesia administrated via a vaporizer for most of the procedure, except the wash-out and infusion periods. Because VA concentration in the brain was equivalent to 0.2–0.3MAC, the rabbits were considered to be hypnotized or sedated during the infusion period [22]. (ii) Given the minimally invasive technique used, airway topical anesthesia and wound local anaesthetics infiltration can provide extra analgesia and enhance the tolerance to pain stimulus [19, 25, 26]. (iii) Rabbits have a natural tolerance for restraint and can hence remain immobile in a confined space [27]. Considering this unique behavior, it is fair to assume that even a partially conscious rabbit can uneventfully undergo a cerebral MRI and abdominal ultrasound [27, 28]. In our study, during the wash-out and infusion periods, instead of being fixed on the bench, the animal was unlocked and wrapped in a surgical towel with slight manual restrain, which not only helped to relieve pain and alleviate stress, but also made the animal immobile. All rabbits stayed immobile and did not display any signs of distress or discomfort during the procedure.

In our study, because of the limited blood supply to the spinal cord, only the thoracolumbar region (below T3), not the entire spinal cord, could be selectively anesthetized during aortic delivery of emulsified isoflurane and sevoflurane. However, our method still has several advantages. Firstly, because of less trauma, the integrity of cerebral and spinal cord circulation remains intact in this rabbit model [6, 7, 29]. Secondly, unlike bypass models that need special and expensive equipment, this rabbit model is simple and less expensive. At the end of the study, all rabbits were awake and no fatalities occurred. This confirmed that infusion of emulsified isoflurane or sevoflurane produced a safe and reversible anesthesia.

In conclusion, a simple method has been successfully established that permits differential delivery of isoflurane and sevoflurane to the spinal cord. This model was found to eliminate 69% isoflurane and 81% sevoflurane from blood via lungs, before acting on the brain. Taking advantage of the unique homosegmental blood supply to the spinal cord in rabbit, this model can also be used to preferentially deliver anesthetics to the thoracic, lumbar or sacro-coccygeal spinal cord, thereby permitting investigation of the effects of anesthetics on different spinal segments.

Relevant data underlying the findings described in manuscript.

(XLS)

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20 Nov 2019

PONE-D-19-27037

Development of a simple method for differential delivery of volatile anaesthetics to the spinal cord of the rabbit

PLOS ONE

Dear Peng Zhang, Yao Li and Ting Xu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewer #1: The methods manuscript by Zhang et al uses a rabbit mode to differentially deliver volatile anesthetics to the brain versus spinal cord using emulsified agents infused into the descending aorta. Although this has been achieved in prior studies using brain bypass, the main advantage of this model is that it is simple and minimally invasive. However the authors statements that prior models suffered from "CNS damage" is not supported at least in prior goat models where MAC values remained normal before and following bypass, or when bypass was implemented and equal amounts of anesthetic were delivered to the brain and spinal cord. Therefore these comments should be omitted or toned down, and rather the authors should highlight the real advantage of being simpler, less labor intensive and less costly.

The reported differences in partial pressures of volatile anesthetics in the femoral vein ("spinal cord") were 3.5 to 5 fold greater than in jugular vein ("brain") which is very good separation similar to bypass models. However, this model is limited in that unlike bypass models, anesthetic cannot be regulated to the brain. THIS PRESENTS A SERIOUS ANIMAL WELFARE CONCERN, especially for VA with low solubility, like sevoflurane used in the present study. That is, with peri-MAC concentrations delivered to the spinal cord, anesthetic concentrations to the brain reach low enough concentrations to permit the rabbits to become conscious during the experiment. This welfare concern and the fact that brain anesthetic cannot be regulated is a major limitation to the model the authors failed to mention.

The minimal invasive techniques makes this less concerning, however it is still concerning. Perhaps if the rabbits were acclimated and trained to lie quietly in a restrainer with the mask on before conducting the procedure, this would eliminate most of the animal welfare issues. However I cannot say if this is within a rabbits behavioural repertoire.

Last, while I am sympathetic to the language barriers non-english speaking groups face, the manuscript should be reviewed and edited by a fluent english writer BEFORE submission. While some parts are written fairly well, I still extensively edited the manuscript (see attached file) for basic syntax, grammar and clarity. It is not appropriate to place this burden on a scientific reviewer, and moreover the authors should have a vested interest in submitting a comprehensible manuscript to maximize chances of being accepted.

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Submitted filename: Zhang etal_emulsified VA_rabbitt_reviewer edit.pdf

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1 Jan 2020

Dr. Solevåg

Submitted filename: Rebuttal letter.docx

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22 Jan 2020

PONE-D-19-27037R1

Development of a simple method for differential delivery of volatile anesthetics to the spinal cord of the rabbit

PLOS ONE

Dear Dr. Xu Ting Xu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR:

Reviewer 1 asks for minor edits. The manuscript will be accepted for publication in PLOS ONE provided that the authors can make these changes:

Overall the authors have addressed comments and concerns, with some minor ones remaining although I feel these do not require re-review:

The manuscript still needs some minor English language editing including the revised text, however it is much improved.

The authors make good points about animal welfare concerns however the comments are mostly speculative without reference to what they actually observed at the relevant timepoints. Please add something to the effect "animals did not display signs of distress or discomfort during the infusion and post-infusion time periods" (assuming this was true).

==============================

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Kind regards,

Anne Lee Solevåg, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Reviewer 1 asks for minor edits. The manuscript will be accepted for publication in PLOS ONE provided that the authors can make these changes:

Overall the authors have addressed comments and concerns, with some minor ones remaining although I feel these do not require re-review:

The manuscript still needs some minor English language editing including the revised text, however it is much improved.

The authors make good points about animal welfare concerns however the comments are mostly speculative without reference to what they actually observed at the relevant timepoints. Please add something to the effect "animals did not display signs of distress or discomfort during the infusion and post-infusion time periods" (assuming this was true).

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Reviewer #1: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Overall the authors have addressed comments and concerns, with some minor ones remaining although I feel these do not require re-review:

The manuscript still needs some minor English language editing including the revised text, however it is much improved.

The authors make good points about animal welfare concerns however the comments are mostly speculative without reference to what they actually observed at the relevant timepoints. Please add something to the effect "animals did not display signs of distress or discomfort during the infusion and post-infusion time periods" (assuming this was true).

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Reviewer #1: No

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30 Jan 2020

We agree with the reviewer and have performed some modifications in our revised manuscript. Three references (reference 25, 26, 28) was added in the Discussion section to support our results. In addition, the sentence “All rabbits stayed immobile and did not display any signs of distress or discomfort during the procedure” was also added (lines 225-227). Furthermore, we have had our revised manuscript edited by a native English-speaking editor to improve the language quality.

Submitted filename: Rebuttal letter revised 2.docx

Click here for additional data file.

3 Feb 2020

Development of a simple method for differential delivery of volatile anesthetics to the spinal cord of the rabbit

PONE-D-19-27037R2

Dear Dr. Ting Xu,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Anne Lee Solevåg, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

10 Feb 2020

PONE-D-19-27037R2

Development of a simple method for differential delivery of volatile anesthetics to the spinal cord of the rabbit

Dear Dr. Xu:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Anne Lee Solevåg

Academic Editor

PLOS ONE