Category Archives: V2I12014

Outcome of Carotid Endarterectomy after Regional Anesthesia versus General Anesthesia A Retrospective Study Using Two Independent Databases

September 4, 2014V2I12014liur

Jiabin Liu, MD, PhD, Hector Martinez-Wilson, MD, PhD, Mark D. Neuman, MD, MSCE, Nabil Elkassabany, MD, MSCE, Edward Andrew Ochroch, MD, MSCE

Department of Anesthesiology and Critical Care, the Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 19104.

Corresponding Author:

Jiabin Liu, MD, PhD, Department of Anesthesiology and Critical Care, the Perelman School of Medicine, University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA, USA 19104. Email: Jiabin.liu@uphs.upenn.edu Phone: 215-573-8239.

Running title: Regional Anesthesia for CEA with Less Complication

Disclosures: The study was supported by departmental fund to JL at the University of Pennsylvania. MDN receives fund from National Institute on Aging (K08AG043548-01). The authors declare no conflict of interest.

NSQIP disclosure: “The American College of Surgeons National Surgical Quality Improvement Program and the hospitals participating in the ACS NSQIP are the source of the data used herein; they have not verified and are not responsible for the statistical validity of the data analysis or the conclusions derived by the authors.”

Keywords: Carotid Endarterectomy; Regional Anesthesia; Outcome; NSQIP; NY-SID

Citation: Jiabin Liu, Hector Martinez-Wilson, Mark D. Neuman, Nabil Elkassabany, Edward Andrew Ochroch. Outcome of Carotid Endarterectomy after Regional Anesthesia versus General Anesthesia A Retrospective Study Using Two Independent Database Trans Periop & Pain Med 2014, 1(2):*****.

Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Abstract

Background: Carotid endarterectomy (CEA) is effective in reducing stroke risk in selected patient groups. The ideal anesthetic technique remains controversial in light of literature between general anesthesia (GA) and regional anesthesia (RA) for CEA.

Methods: We studied the NSQIP data from 2005 to 2012. There were 32,718 patients receiving general anesthesia (GA) and 5,384 patients receiving regional anesthesia, local anesthesia, or monitored anesthesia care (RA). The outcome measurements of 30 days postoperative complications were death, stroke, coma, unplanned intubation, on ventilator > 48 hours, cardiac arrest, and myocardial infarction. We next studied NY-SID data from 2007 to 2011. There were 13,913 patients receiving GA and 3,145 patients receiving RA. The outcome measurements by discharge time were death, stroke, paraplegia, new neurological disorder, aspiration, respiratory failure, pulmonary resuscitation procedure (include intubation), cardiac arrest, cardiac resuscitation procedure, myocardial infarction, and congestive heart failure. All analyses were risk adjusted with propensity score matching algorithm.

Results: There were significant differences in incidences of un-expected intubation (1.21% vs. 0.55%, P=0.001), and myocardial infarction (0.80% vs. 0.35%, P=0.039) between GA and RA respectively in NSQIP data. GA group had significant higher incidences of aspiration (0.61% vs. 0.19%, P=0.014), and pulmonary resuscitation procedure (including intubation) (1.02% vs. 0.54%, P=0.044) than RA group in NY-SID data.

Conclusions: In comparison to GA, patients receiving RA had significant lower risks of postoperative unplanned intubation and/or pulmonary resuscitation procedure after carotid endarterectomy.

Introduction

Carotid endarterectomy (CEA) is effective in reducing stroke risk in selected patient groups. CEA is commonly performed under general anesthesia (GA), regional anesthesia, local anesthesia, or monitored anesthesia care. Based on the typical intraoperative care paradigms, we chose to define regional anesthesia (RA) to include any of the above local anesthetic based anesthesia practice, including regional anesthesia, local anesthesia, and monitored anesthesia care. The choice of anesthesia is largely based on patient factors, surgeon’s preference, and the culture of the institution. The ideal anesthetic technique remains controversial as multiple small studiesproduced conflicting results regarding the association of GA versus RA with mortality(1-7), stroke(2-8), hemodynamic homeostasis (1,3-5), and cardiac morbidity (3,6,7).

The GALA (general anesthesia versus local anesthesia for carotid surgery) study was the only large randomized controlled clinical trial with 3526 patients, and it concluded that there was no difference in incidences of death, stroke, or myocardial infarction between GA and RA (9). While GALA study provided the most convincing comparisons between GA and RA, it has its limitation. The GALA study reported that 65% of the patients were ASA I or ASA II (9), while other study revealed ~90% of patients undergoing CEA were ASA III or IV (10).

The American College of Surgeons National Surgical Quality Improvement Program (NSQIP) is a nationally validated outcome-based program to measure surgical outcomes. It contains 140 variables, including patient demographic information, preoperative comorbidities, intraoperative variables, and 30-day postoperative complications. A retrospective study by Schechter and colleagues on the NSQIP data from 2005 to 2009 looking at the composite risks of stroke, myocardial infarction, and death did not show significant patient outcome differences between GA and RA groups (2.8% versus 3.6%) undergoing CEA (11). However, Schechter et al. reported significant differences in secondary complications between GA and RA (4.1% versus 2.9%) without detail information on the nature of these differences (11). Leichtle et al. studied the same NSQIP data from 2005 to 2009 with a propensity matching strategy, and concluded that GA was associated with higher incidence of myocardial infarction (odds ratio 5.41), while no differences were reported for mortality and stroke risks (10).

With the release of NSQIP data from 2010-2012 we proposed to take advantage of the much larger dataset to study low incidence clinically relevant postoperative complications during CEA. We hypothesized that there are no differences on 30-day postoperative central nervous, pulmonary, and cardiovascular system complications between GA and RA patients.

New York State Inpatient Database is another independent database publically available via the US Agency for Healthcare Research and Quality’s (AHRQ) Health Care Utilization Project (HCUP). The database contains information on patient demographic information, International Classification of Diseases-9-Clinical Modification (ICD-9-CM) code for diagnoses, ICD-9-CM code for procedures, anesthesia type, and discharge status. There was no previous study on outcome differences between GA and RA among CEA patients in the NY-SID data. Hereby we propose to utilize the NY-SID data as a replication set to further test our hypothesis.

Materials and Methods

Data source: This study was exempted by the institutional review board (the University of Pennsylvania, Philadelphia, Pennsylvania, USA 19104).

NSQIP Data:We acquired the American College of Surgeons National Surgical Quality Improvement Program (NSQIP) database from 2005 to 2012 (http://site.acsnsqip.org). NSQIP members prospectively submit data on 140 variables, which are validated via strict standardized protocol. The data include demographic information, pre-existing comorbidities, intraoperative variables, and postoperative complications for 30 days after the surgery. The full list of information collected is available at NSQIP (http://site.acsnsqip.org/participant-use-data-file/). The NSQIP participant user data files included 152,490 subjects in 2005 and 2006, 211,407 subjects in 2007, 271,368 subjects in 2008, 336,190 subjects in 2009, 363,431 subjects in 2010, 442,149 subjects in 2011, and 543,885 subjects in 2012.

NY-SID Data: We acquired the US Agency for Healthcare Research and Quality’s (AHRQ) Healthcare Cost and Utilization Project (HCUP) New York State Inpatient Database (NY-SID) from 2007 to 2011 (http://www.hcup-us.ahrq.gov). NY-SID includes the collection of all encounter-level information in the state of New York. The data include demographic information, anesthesia type, ICD-9-CM diagnosis code, ICD-9-CM procedure code, AHRQ comorbidity measures of various organs/systems, and discharge status. The full list of information collected is available online (http://www.hcup-us.ahrq.gov/db/state/siddist/sid_multivar.jsp, last accessed August 15, 2014). The NY-SID data included 2,608,615 subjects in 2007, 2,629,383 subjects in 2008, 2,661,905 subjects in 2009, 2,612,610 subjects in 2010, and 2,578,680 subjects in 2011.

Study Sample Definition:

NSQIP Study Sample: To define our study cohort in NSQIP data, we included patients with the Current Procedural Terminology (CPT) code for carotid endarterectomy as their principal procedure (CPT code 35301). There were total of 54,450 entries with the listed CPT code as principal procedure. We first removed patients with ICD-9-CM diagnosis other than carotid occlusion and stenosis (ICD-9 433.1, 433.10, or 433.11). We next excluded patients with other significant concurrent procedures as defined by a relevant concurrent CPT code, which could have significantly effects on the choice of anesthesia and postoperative complication rates. (e.g. combined CEA and CABG) We elected to apply work relative value unit >2.11 as the cutoff criteria to eliminate patients with significant concurrent procedures, while maintaining patients with relevant benign procedures that were relevant to carotid endarterectomy (such as angiography, ultrasonography, arterial cannulation, etc.). We then removed entries with more than five missing comorbidity data points. Next, we excluded patients who received anesthesia type other than general, local, regional, or monitored anesthesia care. We also excluded patients with ASA classification 5 (moribund). Last, we excluded patients who had prior operations within 30 days, pneumonia, ventilator dependence, systemic inflammatory response syndrome (SIRS), sepsis, septic shock, or contaminated/infected/dirty wound classification preoperatively. A diagram illustrating the defining process is summarized in figure 1.

NY-SID Study Sample: To define our study cohort in NY-SID data, we included patients with the primary diagnosis of carotid occlusion and stenosis with or without cerebral infarction (ICD-9-CM 433.10 or 433.11). There were total of 25,336 entries. We first removed patients without ICD-9-CM procedure code (3812) in the first three listed procedures. Next, we excluded patients who received anesthesia type other than general, local, or regional anesthesia. Last, we excluded patients who had pneumonia or were ventilator dependent preoperatively. A diagram illustrating the defining process is summarized in figure 2.

Exposure Variable:

The NSQIP Participant User Data File coded anesthesia types into the following categories: general, local, regional, monitored anesthesia care, spinal, epidural, other, none, or unknown. In cases where general anesthesia was used concurrently with other type(s) of anesthesia, patients were coded as receiving general anesthesia. For the present study, we grouped patients receiving local, regional, or monitored anesthesia care together in a single category as regional anesthesia.

The NY-SID codes method of anesthesia types into the following categories: local, general, regional, other, none, or unknown. In cases where general anesthesia was used concurrently with other type(s) of anesthesia, patients were coded as receiving general anesthesia. For the present study, we grouped patients receiving local and regional anesthesia together in a single category as regional anesthesia.

Study Variables:

The NSQIP dataset contains demographic information (age, gender, height, weight, race), type of anesthesia, American Society of Anesthesiologists (ASA) Physical Status Classification, level of functional dependence prior to surgery in activities of daily living, wound classification, and comorbidities. For this study, we created variables corresponding to the individual system or organ: severe chronic obstructive pulmonary disease (COPD), congestive heart failure, coronary artery disease (defined as history of myocardial infarction, prior percutaneous coronary intervention, previous cardiac surgery, or history of angina in one month before surgery), peripheral vascular disease (defined as history of revascularization/amputation for peripheral vascular disease, or rest pain/gangrene), hypertension requiring medications, diabetes mellitus with or without insulin treatment, end stage liver disease (defined as presence of ascites, or esophageal varices), kidney failure (defined as acute renal failure, or currently on dialysis), central nervous system (CNS) disease (defined as impaired sensorium, coma>24 hours, history of transient ischemic attack, cerebrovascular accident/stroke with or without neurological deficit, or tumor involving CNS), spinal cord injury (defined as hemiplegia, paraplegia, or quadriplegia), and active malignancy (defined as disseminated cancer, chemotherapy, or radiotherapy for malignancy).

The NY-SID dataset contains demographic information (age, gender, race), type of anesthesia, ICD-9-CM diagnosis code, diagnosis present on admission indicator, ICD-9-CM procedure code, and AHRQ HCUP comorbidity measures. The comorbidity measures include congestive heart failure, pulmonary circulation disorders, peripheral vascular disease, chronic pulmonary disease, diabetes, liver disease, renal failure, central nervous system disease, malignancy, and etc. The full list is available at http://www.hcup-us.ahrq.gov/db/state/sasddist/sasd_multivar.jsp; last accessed on Aug 15, 2014. The definition of each comorbidity by ICD-9-CM code is available at http://www.hcup-us.ahrq.gov/toolssoftware/comorbidity/Table2-FY12-V3_7.pdf, last accessed on Aug 15, 2014.

Outcome Variables:

We obtained data on six postoperative complications within NSQIP database. These included stroke/CVA, coma > 24 hours, unplanned intubation, on ventilator > 48 hours, cardiac arrest requiring CPR, and myocardial infarction. All six variables were defined as either diagnosed by surgeon or attending physician, or on the basis of pre-defined clinical and laboratory criteria as specified at NSQIP website.

We were able to obtain data on 10 postoperative complications within NY-SID database. These included stroke, paralysis, new neurological disorder, aspiration, respiratory failure, pulmonary resuscitation procedure (including reintubation and extended ventilator support), cardiac arrest, cardiac resuscitation procedure, myocardial infarction, and congestive heart failure. Each variable is identified via ICD-9-CM code algorithms as previously described (12-15), with additional ICD-9-CM coding algorithms on paralysis and new neurological disorder at HCUP website (http://www.hcup-us.ahrq.gov/toolssoftware/comorbidity/Table2-FY12-V3_7.pdf, last accessed on Aug 15, 2014).

Statistical Analysis: A propensity score was calculated with logistic regression modeling that included all study variables. Variables for the NSQIP matching algorithm include age, gender, BMI, race, ASA Status, level of functional status, and all pre-existing comorbidities. Variables for the NY-SID pairing include age, gender, race, and AHRQ comorbidity measures. The propensity score represented the probability of receiving RA for each patient in the range of 0 to 1. The propensity scores were then applied to create two matched groups of GA and RA with the caliper distance of 0.005 without replicates. The matched cohorts were then compared similarly as described above. All data analyses were executed in STATA 12.1 (StataCorp LP, College Station, TX, USA). Fisher’s exact test and chi-square test were used for categorical data. Student T-test and Wilcoxon test were applied for interval data. Statistic significance was defined as P<0.05. Our main focus was to compare individual central nervous, pulmonary, and cardiac system outcomes between GA and RA groups. To compensate the existing differences on patient characteristics and comorbidities between GA and RA groups, we conducted propensity score matching algorithm (described below) to generate two matching groups of GA and RA patients for further analysis.

Results

The cohort from the NSQIP database

We identified 54,450 patients who underwent carotid endarterectomy via CPT code between 2005 and 2012 in the NSQIP database (Figure 1). We excluded 2973 patients with concurrent ICD-9 codes other than 433.1, 433.10, or 433.11 (Carotid artery occlusion and stenosis). We

Figure 1.Creation of study sample with NSQIP database. CPT: Current Procedural Terminology. ICD-9: International Classification of Diseases-9. MAC: Monitored Anesthesia Care. GA: General Anesthesia. (review original figure 1 here)

then removed 1432 entries with major concurrent procedures, and 10,653 entries with more than 5 missing pre-existing comorbidity data fields. We next excluded 492 patients with types of anesthesia other than local, regional, monitored anesthesia care, or general anesthesia. We further eliminated patients with ASA classification 5 – moribund (n=10), unknown ASA classification (n=27), prior operations within 30 days (n=366), preoperative pneumonia (n=45), ventilator dependent (n=6), systemic inflammatory response syndrome (SIRS)/sepsis/septic shock (n=242), and contaminated/infected/dirty wound classification (n=102). The final cohort contained 38,102 patients (Figure 1).

There were 32,718 (85.87%) GA, and 5384 (14.13%) RA subjects in the NSQIP dataset (Table 1). There were statistical, but probably not clinically, significant differences in age, gender, BMI, and race. The average hospitalization days were 2.47 vs. 2.09 days between GA and RA group (P<0.0001).

Table 1. NSQIP Patient Demographic Information Summary by Anesthesia Type

		General Anesthesia			Regional Anesthesia			P-value
		N/Mean		%/SD	N/Mean		%/SD
Total Subjects (N)		32718	85.87%	5384	14.13%
Age:	70.94	±	9.51	72.15	±	9.32	<0.0001
Gender:	Male	19271	59.00%	3243	60.45%	0.046
Female	13393	41.00%	2122	39.55%
Unknown	54	19
BMI:	28.13	±	6.63	27.81	±	6.16	0.0009
Race:	White	27572	90.60%	4502	92.24%	<0.001
Black	1344	4.42%	147	3.01%
Hispanic	988	3.25%	151	3.09%
Others	529	1.74%	81	1.66%
	Unknown	2285		503
LOS	2.47	±	5.31	2.09	±	6.06	<0.0001

All values were reported as mean ± SD, or percentage.

The unit of measure for BMI is kilogram/meter².

The GA group had higher prevalence of ASA class III and IV patients (91.14% vs 90.04%), COPD (10.51% vs 9.51%), diabetes on insulin (9.69% vs 8.64%), and central nervous system disease (43.33% vs 41.18%) than RA group (Table 2). The RA group had more patients with hypertension requiring medication (86.53% vs 85.20%), and spinal cord injury (0.54% vs 0.35%) than GA group. All subjects were then fitted with the propensity score matching analysis, and a subgroup of matched patients (n=4880 per group) from the total cohort was generated for further comparison. Analysis of preoperative demographic information and pre-existing comorbidities indicated covariate balance between GA and RA group (Table 3 and Table 4).

Table 5 lists the incidence of central nervous, pulmonary, and cardiovascular system complications before and after propensity score matching within 30 days postoperatively in the NSQIP data. The overall 30 day mortality was 0.76% and 0.72% between GA and RA subjects respectively (P=0.906). The RA group had lower incidences of unplanned intubation after surgery (0.55% vs 1.21%, P=0.001), and myocardial infarction (0.45% vs 0.80%, P=0.039).

Table 2. NSQIP Prevalence of Pre-existing Comorbidities.

			General Anesthesia		Regional Anesthesia
Comorbidity			N	%	N	%	P-value
ASA classification	4-Life Threat		4201	12.84	568	10.55	<0.001
3-Severe Disturbance		25618	78.30	4280	79.49
2-Mild Disturbance		2850	8.71	533	9.90
1-No Disturbance		49	0.15	3	0.06
Functional health status Prior to Surgery	Dependent		97	0.30	10	0.19	0.388
Partially Dependent		1375	4.21	225	4.18
Independent		31221	95.50	5149	95.64
CHF in 30 days before surgery			258	0.79	41	0.76	0.934
Coronary Artery Disease	11785	36.02	1964	36.48	0.520
Peripheral Vascular Disease	3257	10.01	509	9.45	0.210
HTN requiring medication	27877	85.20	4659	86.53	0.010
Dyspnea	At Rest		346	1.06	52	0.97	0.836
Moderate Exertion		5596	17.10	927	17.22
History of severe COPD	3439	10.51	512	9.51	0.026
End Stage Liver Disease	26	0.08	5	0.09	0.795
Renal Failure	376	1.15	49	0.91	0.141
Diabetes Mellitus	Insulin		3172	9.69	465	8.64	0.047
Oral/Non-insulin		6062	18.53	1005	18.67
Central Nervous System Disease			14178	43.33	2217	41.18	0.003
Spinal Cord Injury	115	0.35	29	0.54	0.042
Disseminated cancer, Chemotherapy/Radiotherapy			148	0.45	27	0.50	0.587
Bleeding disorders		6723	20.55	1083	20.12	0.477

ASA: American Society of Anesthesiologists; CHF: Congestive heart failure; HTN: Hypertension; COPD: Chronic Obstructive Pulmonary Disease.

Table 3. NSQIP Patient Demographic Information Summary by Anesthesia Type in the

Propensity Score Matched Sub-groups (N=4880 per group).

		General Anesthesia			Regional Anesthesia			P-value
		N/Mean		%/SD	N/Mean		%/SD
Age:	72.10	±	9.21	72.16	±	9.36	0.7462
Gender:	Male	3024	61.97%	2956	60.57%	0.164
Female	1856	38.03%	1924	39.43%
BMI:	27.86	±	6.12	27.84	±	6.16	0.8542
Race:	White	4502	92.25%	4501	92.23%	0.253
Black	144	2.95%	147	3.01%
Hispanic	131	2.68%	151	3.09%
Others	103	2.11%	81	1.66%

All values were reported as mean ± SD, or percentage. The unit of measure for BMI is kilogram/meter². “Other race” included patients listed as Native Hawaiian or Pacific Islander, Asian or Pacific Islander, Asian, and American Indian or Alaska

Native in the NSQIP database.

Table 4. NSQIP Prevalence of Pre-existing Comorbidities in the Propensity Score Matched Sub-groups

(N=4880 per group).

			General Anesthesia		Regional Anesthesia
Comorbidity			N	%	N	%	P-value
ASA classification	4-Life Threat		520	10.66	528	10.82	0.381
3-Severe Disturbance		3927	80.47	3877	79.45
2-Mild Disturbance		432	8.85	472	9.67
1-No Disturbance		1	0.02	3	0.06
Functional health status Prior to Surgery	Dependent		7	0.14	7	0.14	0.975
Partially Dependent		201	4.12	205	4.20
Independent		4672	95.74	4668	95.66
CHF in 30 days before surgery			34	0.70	38	0.78	0.723
Coronary Artery Disease	1766	36.19	1793	36.74	0.585
Peripheral Vascular Disease	449	9.20	458	9.39	0.780
HTN requiring medication	4241	86.91	4228	86.64	0.720
Dyspnea	At Rest		52	1.09	50	1.02	0.951
Moderate Exertion		843	17.27	839	17.19
History of severe COPD	466	9.55	467	9.57	1
End Stage Liver Disease	9	0.18	5	0.10	0.424
Renal Failure	50	1.02	41	0.84	0.400
Diabetes Mellitus	Insulin		421	8.63	430	8.81	0.873
Oral/Non-insulin		898	18.40	912	18.69
Central Nervous System Disease			1967	40.31	2015	41.29	0.333
Spinal Cord Injury	25	0.51	24	0.49	1
Disseminated cancer, Chemotherapy/Radiotherapy			30	0.61	26	0.53	0.688
Bleeding disorders		982	20.12	1000	20.49	0.669

ASA: American Society of Anesthesiologists; CHF: Congestive heart failure; HTN: Hypertension;

COPD: Chronic Obstructive Pulmonary Disease.

Table 5. NSQIP Incidences of 30-days Post-operative Complications before and after Propensity Score Matching

		Before Propensity Score Matching							After Propensity Score Matching
	GA(N=32718)				RA(N=5384)				GA(N=4880)		RA(N=4880)
Variable Label	N		%	N		%	P-value	N		%	N	%	P-value
Mortality	238		0.73	37		0.69	0.795	37		0.76	35	0.72	0.906
Stroke/CVA	481		1.47	77		1.43	0.854	76		1.56	74	1.52	0.934
Coma >24 hours	20		0.06	5		0.09	0.387	1		0.02	5	0.10	0.219
Unplanned Intubation	365		1.12	27		0.50	<0.001	59		1.21	27	0.55	0.001
On Ventilator > 48 Hours	212		0.65	24		0.45	0.091	38		0.78	23	0.47	0.071
Cardiac Arrest Requiring CPR	84		0.26	10		0.19	0.377	9		0.18	10	0.20	1
Myocardial Infarction	257		0.79	25		0.46	0.010	39		0.80	22	0.45	0.039

GA: General anesthesia; RA: Regional anesthesia; CVA: Cerebrovascular accident; CPR: Cardiopulmonary resuscitation.

Table 6. NY-SID Patient Demographic Information Summary by Anesthesia Type.

		General Anesthesia			Regional Anesthesia			P-value
		N/Mean		%/SD	N/Mean		%/SD
Total Subjects (N)		13913	81.56%	3145	18.44%
Age:	71.49	±	9.43	71.71	±	9.52	0.2433
Gender:	Male	8009	57.56%	1889	60.06%	0.010
Female	5904	42.44%	1256	39.94%
Race:	White	11942	86.52%	2797	89.22%	<0.001
Black	467	3.38%	58	1.85%
Hispanic	755	5.47%	107	3.41%
Others	638	4.62%	173	5.52%
LOS	2.60	±	4.91	2.05	±	3.14	<0.001

All values were reported as mean ± SD, or percentage. “Other race” included patients listed as

Asian or Pacific Islander, Native American, or other in the NY-SID database. LOS: Length of

Hospital Stay in days.

Table 7. NY-SID Patient Demographic Information Summary by Anesthesia Type in the

Propensity Score Matched Sub-groups (N=3134 per group).

		General Anesthesia			Local Anesthesia			P-value
		N/Mean		%/SD	N/Mean		%/SD
Age:	71.62	±	9.39	71.70	±	9.53	0.7224
Gender:	Male	1883	60.08%	1883	60.08%	1
Female	1251	39.92%	151	39.92%
Race:	White	2779	88.67%	2796	89.22%	0.916
Black	63	2.01%	58	1.85%
Hispanic	112	3.7%	107	3.41%
Others	180	5.4%	173	5.52%

All values were reported as mean ± SD, or percentage. “Other race” included patients listed as

Asian or Pacific Islander, Native American, and other in the NY-SID database.

Table 8. NY-SID Prevalence of Pre-existing Comorbidities in the Propensity Score Matched

Sub-groups (N=3134 per group).

	General Anesthesia		Regional Anesthesia
Comorbidity	N	%	N	%	P-value
Alcohol	49	1.56	48	1.53	1
Deficiency Anemia	117	3.73	135	4.31	0.274
Rheumatoid Arthritis / Collagen Vascular Disease	48	1.53	46	1.47	0.917
Chronic Blood Loss Anemia	9	0.29	10	0.32	1
Congestive Heart Failure	140	4.47	162	5.17	0.215
Chronic Pulmonary Disease	642	20.49	657	20.96	0.663
Coagulopathy	21	0.67	25	0.80	0.658
Drug Abuse	3	0.10	6	0.19	0.507
Hypertension	2563	81.78	2564	81.81	1
Liver Disease	25	0.80	29	0.93	0.682
Fluid / Electrolyte Disorders	73	2.33	87	2.78	0.298
Obesity	185	5.90	196	6.25	0.597
Paralysis	0	0	4	0.13	0.125
Peripheral Vascular Disorders	560	17.87	574	18.32	0.670
Pulmonary Circulation Disorders	32	1.02	34	1.08	0.902
Renal Failure	219	6.99	238	7.59	0.382
Valvular Disease	231	7.37	246	7.85	0.505
Weight Loss	4	0.13	8	0.26	0.387
Mental Disorder	199	6.35	213	6.80	0.508
Cancer	43	1.37	49	1.56	0.600
Diabetes	922	29.42	938	29.93	0.678

Mental disorder: includes depression and psychoses; Cancer: includes lymphoma, metastatic

Cancer, and solid tumor without metastasis; Diabetes includes insulin dependent and insulin

independent diabetes;

Table 9. NY-SID Prevalence of Pre-existing Comorbidities.

			General Anesthesia		Regional Anesthesia
Comorbidity			N	%	N	%	P-value
Alcohol	163	1.17	48	1.53	0.108
Deficiency Anemia	810	5.82	135	4.29	0.001
Rheumatoid Arthritis/ Collagen Vascular Disease			313	2.25	48	1.53	0.011
Chronic Blood Loss Anemia	34	0.24	11	0.35	0.333
Congestive Heart Failure	749	5.38	163	518	0.693
Chronic Pulmonary Disease			2987	21.47	660	20.99	0.563
Coagulopathy	139	1.00	25	0.79	0.313
Drug Abuse	49	0.35	6	0.19	0.167
Hypertension	11444	82.25	2573	81.81	0.553
Liver Disease	89	0.64	29	0.92	0.095
Fluid / Electrolyte Disorders	711	5.11	87	2.77	<0.001
Obesity			771	5.54	196	6.23	0.135
Paralysis	27	0.19	4	0.13	0.642
Peripheral Vascular Disorders			2405	17.29	575	18.28	0.185
Pulmonary Circulation Disorders			137	0.98	34	1.08	0.620
Renal Failure	1022	7.35	239	7.60	0.624
Valvular Disease	1023	7.35	247	7.85	0.328
Weight Loss	32	0.23	8	0.25	0.838
Mental Disorder	987	7.09	215	6.84	0.643
Cancer			209	1.50	49	1.56	0.808
Diabetes		4475	32.16	939	29.86	0.012

Mental disorder: includes depression and psychoses; Cancer: includes lymphoma, metastatic Cancer,

and solid tumor without metastasis; Diabetes includes insulin dependent and independent diabetes.

The cohort from the NY-SID data

We then set out to replicate these findings in the NSQIP data in the NY-SID data where we identified 25,336 patients with CEA listed within the first three procedures (Figure 2). We then removed 1863 patients without carotid occlusion and stenosis with or without cerebral infarction enlisted as primary diagnosis. Next, we excluded 6295 patients who received anesthesia type other than general, local, or regional anesthesia. Last, we excluded patients with preoperative pneumonia (n=4) and respiratory failure (n=116). The final cohort contained 17,058 subjects (Figure 2).

There were 13,913 subjects in the GA group and 3,145 subjects in the RA group in the NY-SID patient’s database (Table 6). The average length of hospitalization was 2.60 vs 2.05 days between GA and RA group (P<0.001). Further analysis of the comorbidities indicated several differences (Table 7). The GA group had higher prevalence of patients with anemia (5.82% vs 4.29%), rheumatoid arthritis / collagen vascular disease (2.25% vs 1.53%), fluid / electrolyte

Figure 2.Creation of study sample with NY-SID database. ICD-9: International Classification of Diseases-9. ICD-9-CM: International Classification of Diseases-9 Clinical Modification. GA: General Anesthesia. (review original figure 2 here)

disorder (5.11% vs 2.77%), and diabetes (32.16% vs 29.86%) than RA group. All subjects were then fitted with the propensity score matching analysis with NY-SID variables, and a subgroup of matched patients (n=3134 per group) from the total cohort was generated for further comparison. Analysis of preoperative demographic information and pre-existing comorbidities indicated the equality between GA and RA group (Table 8 and Table 9).

Table 10. NY-SID Incidences of Post-admission Complications before and after Propensity Score Matching

		Before Propensity Score Matching									After Propensity Score Matching
		GA(N=13913)			RA(N=3145)				GA(N=3134)			RA(N=3134)
Variable Label	N		%	N		%	P-value	N		%		N	%	P-value
Mortality	43		0.31	3		0.10	0.035	8		0.26		3	0.10	0.226
Stroke	33		0.24	5		0.16	0.531	4		0.13		5	0.16	1
Paralysis	62		0.45	7		0.22	0.086	12		0.38		7	0.22	0.359
Other Neurologic Disorder	66		0.47	10		0.32	0.299	13		0.41		10	0.32	0.677
Aspiration	73		0.52	6		0.19	0.012	19		0.61		6	0.19	0.014
Respiratory Failure	302		2.17	40		1.27	0.001	49		1.56		40	1.28	0.393
Pulmonary Resuscitation Procedure	178		1.28	17		0.54	<0.001	32		1.02		17	0.54	0.044
Cardiac Arrest	284		2.04	57		1.81	0.438	54		1.72		57	1.82	0.848
Cardiac Resuscitation Procedure	31		0.22	4		0.13	0.384	2		0.06		4	0.13	0.687
Myocardial Infarction	134		0.96	19		0.60	0.059	26		0.83		19	0.61	0.370
Congestive Heart Failure	76		0.55	14		0.45	0.586	10		0.32		13	0.41	0.667

GA: General anesthesia; RA: Regional anesthesia;

The overall inpatient mortalities were 0.26% and 0.10% in the GA and RA groups (P=0.226), which were lower than the 30 days mortality rate in NSQIP data as expected (Table 10). RA group was associated with lower incidences of aspiration (0.19% vs 0.61%, P=0.014), pulmonary resuscitation procedure including reintubation and ventilator support after surgery (0.54% vs 1.02%, P=0.044). There was no difference of myocardial infarction (0.61% vs 0.83%, P=0.370) in the NY-SID data.

Discussion

Our study using prospectively collected NSQIP data of 38,102 CEA patients and NY-SID data of 17,058 CEA patients suggests that regional anesthesia was associated with better outcome indicated by some of the complication indexes.

Why the current study is needed?

There were many studies comparing general anesthesia and regional anesthesia in CEA. However, most studies were limited by sample size to be conclusive. Meta-analysis could potentially draw conclusions to this debate. However, the heterogeneity of these studies could not adequately power the meta-analysis to draw convincing conclusion. A meta-analysis with 48 studies in 2007, including 14 prospective and 34 retrospective studies (16) concluded that, lower incidences of death, stroke, and myocardial infarction in patients receiving RA despite of the limited study power due to the low number of prospective studies (16). However, the multicenter randomized prospective control trial, GALA trial with total of 3526 patients, found no differences in mortality, stroke, myocardial infarction, or length of hospital stay (9). A recent meta-analysis of 14 trials and 4596 operations noticed there were lower incidences of stroke and mortality in RA group compared to GA group, while the differences were not statistically significant (17). We took advantage of the two available large databases to address this debate on anesthesia type and surgical outcome in CEA.

Our results support these previous reports that there were no differences in mortality and stroke risks between GA and RA groups. NSQIP data showed significant lower incidence of myocardial infarction. However, NY-SID data did not support the hypothesis of lower incidence of myocardial infarction. This is likely due to the fact that NY-SID database only included ICD-9-CM code information to the point of hospital discharge, and thus potentially missed later onset complications, such as myocardial infarction, which were routinely monitored in the NSQIP data collection process. Nonetheless, our analysis on two large databases with 38,102 and 17,058 CEA operations provided valuable information on incidence of myocardial infarction at a much larger scale.

Pulmonary complications have not been well studied. Our analysis indicated differences in risks of unplanned intubation after surgery between GA and RA in the NSQIP data. Our analysis also verified the lower incidence of unplanned intubation/ prolonged ventilator support in the RA group in the NY-SID data. Furthermore, NY-SID data indicated RA group is associated with lower incidence of aspiration risk. Unfortunately, the incidence of aspiration could not be studied in the NSQIP data due to lack of such information. To our best knowledge, this is the first study with special focus on pulmonary complications. While the overall mortality and stroke risks were similar between GA and RA patients, these secondary postoperative complications could have significant implications on the requirement of perioperative resources, the length of hospitalization, and the quality of life of patients. The difference might cast significant socioeconomic influence on the patient and health care system.

Limitations of the study

The authors acknowledge that the conclusion of this study is limited due to the retrospective nature of this study design. The coding system of anesthesia type in the NSQIP and NY-SID databases is also a significant limitation. In case of concurrent use of general anesthesia with any other type(s) of anesthesia, patients were coded as receiving general anesthesia. Therefore, patients who were initially planned for RA and converted to GA might represent intraoperative complications that would show up as postoperative complications and thus be miss-assigned with anesthesia type. The NSQIP database also removed hospital and surgeon identification information in order to comply with participation agreement between NSQIP and participating sites. However, this information might be of interest to adjust relative risks. Similarly, there are limitations in the NY-SID database. NY-SID contains encounter-level information, and the integrity of the data relies on the accuracy of ICD-9-CM coding. Although many studies have validated the reliability of ICD-9-CM coding algorithm in outcome studies, the retrospective nature limited the study power.

Conclusion

Our study showed that regional anesthesia was associated with lower incidences of unexpected intubation and pulmonary resuscitation procedure after CEA compared to general anesthesia. The study of two large independent databases, NSQIP database and NY-SID database, provided more evidence on the potential beneficial effect of regional anesthesia on pulmonary complications among CEA patients. However, the choice of type of anesthesia for CEA should also be based on surgeon’s recommendation and patient’s preference considering the limited benefit with regional anesthesia.

Acknowledgement: The authors would like to thank Dr. Stanley Muravchick for reading the manuscript and valuable feedback.

References

Salvinorin A decreases mortality and improves neurological outcome in a neonatal mouse hypoxia model

September 4, 2014V2I12014liur

Chunhua Chen^1,*, Xu Cui^{1, 2*}, Felipe Matsunaga¹, Jingyuan Ma¹, Nan Ma¹, Ted Abel³, Renyu Liu^1,#

¹Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104

²Department of Anesthesiology, Beijing Tongren Hospital, Capital Medical University, Beijing, China 100730

³Departent of Biology, University of Pennsylvania, Philadelphia, PA 19104

*contributed equally as co-first author

Disclosure of funding: This research was supported by NIH K08-GM-093115 (PI: RL) and funding from The Institute for the Translational Medicine and Therapeutics at the University of Pennsylvania (PI, RL and TA).

Running Title: Salvinorin A and neurological outcome after hypoxia

^#Corresponding author: Renyu Liu, M.D., Ph.D. Assistant Professor, Mailing address: Department of Anesthesiology and Critical Care, University of Pennsylvania School of Medicine, 336 John Morgan building, 3620 Hamilton Walk Philadelphia PA, 19104 USA.

Phone : 2156623750, Fax: 2153495078

Email: liur@uphs.upenn.edu

Citation: Chunhua Chen,*Xu Cui,* Felipe Matsunaga1, Jingyuan Ma, Nan Ma, Ted Abel, Renyu Liu. Salvinorin A decreases mortality and improves neurological outcome in a neonatal mouse hypoxia model. Transl Perioper Pain Med 2014, 1(2):*****.

Publication date: September 8^th, 2014.

Abstract

Neonatal hypoxic insults induce a variety of problems that include significant neonatal morbidity and long-term neurological deficits, such as motor deficits and learning disorders. Our previous study demonstrated that salvinorin A (SA), a non-nitrogenous selective kappa opioid agonist, protects the brain following ischemic/reperfusion injury in piglets. Here, we studied the neurological outcome after hypoxic insult in the presence and absence of SA pretreatment in neonatal mice. Eighty-three C57BL/6J mouse pups (postnatal day 1) were randomly divided into the following 3 groups: a control group (G1, n=11) received i.p. injection of DMSO (17%, same as in other groups); a hypoxia group (G2, n=46) received DMSO immediately before hypoxic insult; and a SA group (G3, n=26) received i.p. injection of 0.5 mg/kg SA in DMSO immediately before hypoxic insult. The hypoxic insult was induced by placing the pups in a sealed chamber with a mixture gas with 8% oxygen and 92% nitrogen for 120 minutes. Mortality rate in each group was determined. From postnatal day 2 to postnatal day 21, the pups were weighed and developmental motor behaviors (crawling, walking, running, head point and sniffing, sitting, rearing and eye opening) were observed for 3 minutes every day. Placing reflex, cliff aversion, negative geotaxis test, righting response and forelimb grasping tests were also performed. The first appearance of the neurodevelopmental milestone that continued for two consecutive days was determined. The long-term neurobehavioral effects of SA were observed at the age of 10-11 weeks by utilizing zero maze, Barnes maze and fear conditioning testing. Data were presented as % or mean ± SEM. SA significantly decreased the mortality from 70% to 38.5% (p=0.0135) in hypoxia group. Meanwhile, body weight on postnatal day 2 and day 3 elevated in SA treated group (p=0.0318 and p=0.0221, respectively) compared to that in the hypoxia group. Furthermore, the SA group presented better performance of emergency of rearing at 18.7 ± 0.4 days compared to 19.9 ± 0.3 days in the hypoxia group (p=0.0252). Meanwhile, SA rescued hypoxia-induced delays in forelimb grasping, cliff aversion, righting response, eye opening and rearing activity. No statistically significant differences for the neurological outcomes tested in this study beyond 21 days were observed among groups. The reduction in mortality and improvement of the short-term neurological outcome by SA in a neonatal mice hypoxia model suggested its potential use as a medication to reduce hypoxia-induced neurological injury in the perinatal period.

Keywords: Salvinorin A; hypoxic; neurological outcome; mouse; perinatal

Introduction

Neonatal hypoxic-ischemic (HI) injury can induce high mortality and lifelong catastrophic neurologic and neuro-developmental deficits that include epilepsy, learning disabilities, and behavioral disorders (1). Unfortunately, no effective drug is available for managing HI-related neurological deficits. Therapeutic hypothermia along with supportive treatment is considered to be the only effective approach (2). However, well-controlled hypothermia can only be applied to highly selected patient populations in well-established facilities and it can only produce an outcome improvement of only about 30% in asphyxiated infants (3). It requires extensive training and can be dangerous(4). Thus, there is a significant medical need to develop a novel medication and/or easily manageable therapeutic strategy to reduce neuronal injury from cerebral HI in the perinatal period.

Salvinorin A (SA) is a highly selective and potent kappa opioid receptor (KOR) agonist extracted from Salvia divinorum, a plant that has been consumed by humans for several centuries (5). Using a piglet model, we demonstrated that SA administration before or after brain HI protects the brain from HI and can dilate cerebral vessels significantly, which was an important feature needed during brain hypoxia (6-8), indicating that SA could be a potential medication for brain protection for neonates. Unlike other opioid KOR agonists, SA does not produce frank dysphasia (9) and its intrinsic characteristics make it an alternative therapeutic candidate for various neurological conditions (10-12). These characteristics include: rapid onset, easy passage to the central nervous system, anti-nociceptive and sedative effects, no negative pathological changes in organs following prolonged or high dose exposure, and no respiratory depressive effect (10,12,13). Although the compound has been reported to have hallucinogenic activity, such effects are short-lived and do not induce significant blood pressure and heart rate changes or long term cognition impairment (14). In a recent human study, no persisting adverse effects related to SA were observed (15). Data from these clinical trials indicated that SA could potentially be used in humans safely for therapeutic purposes.

To demonstrate that SA could be potentially used in human subjects, similar effects have to be observed in a second species in addition to the findings in piglet. In the present study, we hypothesized that SA administration could improve outcomes in a neonatal mouse hypoxia model. The mouse model was chosen due to the availability of well-established neurobehavioral testing strategies and the capability to investigate both short-term and long-term neurological outcomes.

Materials and Methods

Salvinorin A (purity ≥98%) was obtained from Apple Pharms (Asheville NC, USA). All other chemicals (reagent grade) were obtained from Sigma-Aldrich (MO, St. Louis, MO, USA).

Experimental protocol

The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania and carried out in accordance with National Institutes of Health guidelines for the use of animals. C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and inbreed pups were used for subsequent experiments. Pups were housed under a 12-hour light/dark cycle after birth with free access to food and water throughout the study. Pups from different litters were divided into 3 groups randomly. Pups in the control group (n=11) received i.p. injection of 17% dimethyl sulfoxide (DMSO) without hypoxic insult; the hypoxia group (n=46) received i.p. injection of 17% DMSO before hypoxic insult; SA group (n=26) received i.p. injection of 0.5mg/kg SA in 17% DMSO before hypoxic insult. Pups of low weight (<1.2g) were excluded from the study. All the neurological tests were performed in a blinded manner.

Hypoxic insult

The hypoxic insult was induced on postnatal day 1. After i.p. injections, pups were put into a glass chamber in a water bath where the temperature was maintained at 37°C. The chamber was tightly closed and filled with 8% oxygen with balanced nitrogen. Following 120 minutes of hypoxic gas exposure, the chamber was opened and the pups were exposed to the air. Chest compressions and limb stretches were performed for up to 20 minutes to regain spontaneous breathing. The pups that successfully regained spontaneous breathing were then returned to their mothers after recovery for 30 minutes.

Mortality rate determination

The neonate mouse will be considered as non-survival if no spontaneous breathing can be restored for 20 min resuscitation immediately after hypoxic insult. The mortality rate will be calculated as: survival rate = (number of mice in a group-number of non-survival)/number of mice in a group.

Short-term developmental motor behavior observation

From postnatal day 2 to postnatal day 21, the pups were weighed and motor behavior observation was performed every day. Pups were observed individually in a plastic testing box. Each of the following behaviors was recorded for 3 minutes: crawling, walking, running, head point and sniffing, sitting, rearing and eye opening. After the observation, placing reflex, cliff aversion, negative geotaxis, righting response and forelimb grasping testing were performed. The first appearance of the behavior that continued for two consecutive days was recorded.

Long-term neurobehavioral effects of SA

The open field testwas performed to gauge locomotor activity on postnatal day 21 at which the mouse was considered juvenile. Mice were individually placed in a 41cm (L) × 41 cm (W) × 30cm (H) plastic box. The “central area” was defined as a 20.5cm x 20.5cm square in the center of the box. The rest of the area was defined as the “peripheral area” (see Figure 5A). The time each mouse spent on exploring the central and periphery parts and the number of rearing behaviors in each respective area were recorded for the first 5 minutes and 30 minutes (16).

At the age of 10-11 weeks, all mice were tested for several behavioral tasks in the order of: zero mazes, barnes maze and fear conditioning. These tasks were performed to examine the basal anxiety level, spontaneous locomotor activity, motor learning, spatial learning and associative memory of the mice. The mice were given 5 days for rest between each behavioral test.

Zero Maze (anxiety-like behavior): “Time in open” measures the anxiety level of mice due to their tendency to avoid open spaces. The anxiety was measured by recording the time spent in the open vs. enclosed space. Increased anxiety correlates to the decreased time in the open spaces. Open/Closed transitions measure the overall activity. Increased transitions equate to greater activity (17).

Barnes Maze (spatial learning): Barnes Mazemeasures the ability to learn with visual cues. “Time to Target” measures the time taken to find the target hole in an arena with 19 other holes and several visual cues. The Barnes Maze is easier to set-up and probably less stressful and a valid alternative to the Water Maze to study spatial memory (18). One of the advantages of the Barnes maze task was that it was not influenced by stress as much as other similar tasks and no strong aversive stimuli or deprivation were used.

Fear Conditioning (memory): Training trial freezing, short term contextual freezing, long term contextual freezing and cued trial freezing were recorded to examine and assess the memory deficits after hypoxia injury (19).

Statistical analyses

Mortality of pups in different groups was compared using a 2×2 contingency table. P-value was calculated with the Fisher exact test. For motor behavior observation, the first days of appearance for each parameter were compared with one-way ANOVA followed by Turkey’s test. Statistical analyses were performed with Graph-Pad Prism (Version 5.0). Data were showed as mean ± SEM. A value of p<0.05 was considered to be statistically significant.

Results

SA significantly decreased mortality

14 out of 46 pups in the hypoxia group and 16 out of 26 pups in the SA group survived after hypoxic injury. SA significantly decreased the mortality rate from 70% to 38.5% (p=0.014) (Figure 1).

Figure 1. The mortality of different groups. The mortality of HP and SA group was 70% and 38.5%, respectively. There was no death in the control group. The difference between SA and HP groups indicated that SA statistically decreased the mortality significantly (*p=0.014, SA+HP vs. HP. SA: Salvinorin A; HP: Hypoxia).

SA increased body weight on postnatal day 2 and day3

SA administration increased body weight compared with those in the hypoxia group on postnatal day 2 (P2) (p=0.0318) and day 3 (P3) (p=0.0221). However, no significant difference was observed among groups on P7, P14 and P21 (Figure 2).

Figure 2. SA elevated the body weight gaining on postnatal day 2 and day 3. Body weight of pups in control, hypoxia, and SA treated groups on P1, P2, P3, P7, P14, and P21 were recorded in our study. SA treatment elevated the body weight gaining on postnatal day 2(P2) (p=0.0318) and postnatal day3 (P3) (p=0.0221) compared to day. However, on P3, P7, P14 and P21, no significant difference was observed between different groups (data not shown) ( *p<0.05 SA+HP vs. HP. P: postnatal; SA: Salvinorin A; HP: Hypoxia).

SA improved developmental outcomes

Pups in the hypoxia group showed delayed appearance of forelimb grasping (day 8.6 ± 1.0), cliff aversion (day 3.4 ± 1.0), righting response (day 8 ± 0.8) and eye opening (day 14.4 ± 0.8). SA administration prevented these delays (day 7.8 ± 0.9, 2.8 ± 0.9, 7.3 ± 0.9, and 13.8 ± 0.6 respectively) (Figure 3).

Figure 3. SA improved some of the developmental neurological outcomes. Hypoxia induced significant delay in forelimb grasping (A), cliff aversion (B), righting response (C), and eye opening (D). SA rescued such hypoxia induced neurological outcomes significantly (^#p<0.05 HP vs. Control ; *p<0.05 SA+HP vs. HP. SA: Salvinorin A; HP: Hypoxia).

Hypoxic pups showed delayed appearance of rearing without support (Figure 4I) at day 19.9±0.3 compared to day 18.2 ± 0.4 in control group (p=0.0034). Hypoxic pups with SA administration presented significant improved performance than hypoxic pups at day 18.7±0.4 (p=0.0252), which is similar to the control group. However, there was no statistical significance with other developmental parameters such as crawling (Figure 4A), walking (Figure 4B), running (Figure 4C), head point and sniffing (Figure 4D), sitting (Figure 4E), rearing with support (Figure 4F)negative geotaxis test (Figure 4G) and placing reflex (Figure 4H), though hypoxic pups also showed delayed appearance compared to both the control and SA groups.

Figure 4. SA showed no statistical improvement on some of the development parameters. There are no statistical significance on some of the development parameters such as walking (A), crawling (B), running (C) , head point and sniffing (D), sitting (E), rearing with support (F), negative geotaxis test (G) and placing reflex (H), though hypoxic pups also showed delayed appearance compared to control and SA groups. Hypoxia pups showed delayed appearance of rearing without support at day 19.86±0.29 compared to day 18.18±0.4435 in control group. Hypoxic pups treated with SA presented better performance than hypoxic pups at day 18.69±0.3843(I), which is similar to the control group (^#p<0.05 HP vs. Control ; * p<0.05 SA+HP vs. HP. SA: Salvinorin A; HP: Hypoxia).

SA did not change long-term neurobehavioral outcome

Although mice in SA group spent more time in the central area during open field testing at P21, this difference was not statistically significant (Figure 5). Meanwhile, the rearing test at P21 in the first 5 minutes and 30 minutes was impaired after hypoxia. This impairment was not observed with SA administration, similar to that in the control group, suggesting that SA could improve hypoxia induced the neurological outcomes (p=0.0167 and p=0.0203, respectively) (Figure 6).

Figure 5. SA did not improve the outcome of open field test significantly. (A) The left figure showed the sketch of the open field test. Mice were individually placed in a 41cm (L) x 41 cm (W) x 30cm (H) plastic box. “Central part of the area” was defined as a 20.5cm x 20.5cm square in the center of the box. The rest of the area was defined as “peripheral part of the area”. The time each mouse spent on exploring the central part and periphery part of the area and the number of rearing behavior were recorded respectively in the first 5 minutes and 30 minutes. (B)The seconds in the central field was recorded in open field test at P21 which showed that HP significantly decreased the seconds compared to the control and SA administration increased seconds in the central filed after hypoxia, but the difference was not statistically significant (^#p<0.05 HP vs. Control. P: postnatal; SA: Salvinorin A; HP: Hypoxia).

Figure 6. SA improved rearing activity at P21 significantly. Rearing test at P21 in the first 5 min and 30 min was impaired by hypoxia and such impairment was not observed with SA administration which was similar to the control group, indicating that SA could improve some of the hypoxia induced long term neurological deficit (^#p<0.05 HP vs. Control ; * p<0.05 SA+HP vs. HP. P: postnatal; SA: Salvinorin A; HP: Hypoxia).

The effects of SA on the anxiolytic properties after hypoxia were tested using an elevated zero maze. There were no significant differences in the percentage of time spent in the open arm and open/closed transitions of the maze between SA and hypoxia groups (Figure 7A, B). The Barnes maze task was used to assess spatial memory deficits (20). However, there was no significant difference among groups (Figure 7C and D). Also, tests of fear conditioning showed that all groups learned at similar rates and no significant differences were observed among groups either (Figure 8).

Figure 7. SA did not improve the long term anxiety level and spatial memory. As for the elevated zero maze to detect the anxiety level and locomotor activity, there were no significant differences in the percentage of time spent in the open arm (A) and open/closed transitions (B) of the maze between SA administration and hypoxia alone groups .The Barnes maze task was used to assess spatial reference memory. In our study, there was no significant difference between the groups of the time to target (C) and time in target region (D). There was also no difference between the control and HP groups (SA: Salvinorin A; HP: Hypoxia).

Figure 8. SA did not change the long term memory deficit. All groups learned at similar rates and there was no significant difference in the long-term behavior of fear conditioning test. Control group showed no impairment compared to HP group in the training trial (A), cued trial (B), short term (C) and long term (D) contextual freezing behavior (p > 0.05). Pretreatment with SA did not change the fear memory compared with HP and Control mice (p > 0.05; SA: Salvinorin A; HP: Hypoxia).

Discussion

In the current study, we found that SA administration before hypoxia reduces the mortality rate and improves several short term neurological development parameters within postnatal 21 days. However, no significant differences for neurological outcomes beyond 21 days were observed among groups. The findings in the present study were consistent with our previous study in piglets which revealed that SA could reduce HI induced injury (6-8).

SA reduced mortality after hypoxia insult

The reduction of the mortality rate is one of the most striking findings in this study. While it could be attributed to the neurological protective effects of SA, it is possible that SA could offer protective effects in other vital organs like heart during hypoxic insults. It has been demonstrated that KOR agonist could protect heart from ischemia (21). Further well defined and targeted studies using SA as a potential therapeutics are needed for future studies. The mortality in the control group for this model was very high (70%), which is higher than that was reported using the modified Rice–Vannucci model (22,23). A possible explanation may be related to the use of the pups at postnatal day 1 in our study instead of postnatal day 10 which was used in the Rice-Vannucci model. However, the feasibility of using hypoxia instead of HI model on pups before postnatal day 10 was supported by previous studies (24,25). Due to the high mortality rate, we remained the design of the study relatively simple for neurological outcome observation only without dose-response studies and therapeutic time window investigation or mechanism studies.

Short-term developmental outcome

Neurodevelopment was impaired by hypoxic insult as indicated by the deficits in neurological developmental behaviors and reflexes (righting, placing, negative geotaxis and cliff aversion). SA improved some of the developmental behaviors (forelimb grasping, eye opening and rearing without support) and some of the reflexes (righting and cliff aversion). Although some significant improvements were observed, SA did not improve all aspects of the developmental parameters that are related to various regions of the brain, which may be due to type II errors or different sensitivity to hypoxic injury in different brain regions.

Long-term neurological outcome

To gauge brain function and the influences on emotional behaviors of SA in the long-term at 10-11 weeks (juvenile age), we chose zero mazes to measure the anxiety level, barnes maze to measure spatial and visual learning, and fear conditioning to measure associative memory. Interestingly, there was not any significant difference among groups. Most importantly, no difference of long-term neurological outcome was found between control and hypoxia group. This might be partially due to the fact that all the survived animals could be more resistant to hypoxic insult, thus there would be lack of sufficient injury to induce long term neurological outcome. For this reason, we did not exam histopathological outcome after the long term behavior outcome test. Although previous reports indicated that SA impaired long-term memory without affecting short-term memory (26), there wasn’t any memory or learning disability observed after SA administration in this study.

Limitations

Firstly, both genders were used in this study. SA distribution and elimination differs among different genders and the kinetics are slower in female than in male monkeys (27), suggesting that gender specific studies should be planned in the future. Secondly, SA was administered before hypoxic insult which was less translational in most of the clinical scenarios except during difficult delivery which was highly predictable that perinatal hypoxia/ischemia is highly likely to occur. Thirdly, this is purely observational study and no mechanism is investigated.

Future directions

The mechanism involved in the protective effects of SA on hypoxic neurons, astrocytes or other cell types still needs to be determined. While it is clear that SA activates KOR, several studies suggested that SA might induce brain metabolic changes in different brain regions through non-KOR dependent pathway (28,29). It is unclear whether such metabolic changes in different brain region could play important role in neurological protective effects.

Inhalation of SA is considered to be one of the efficient ways to obtain its psychoactive effects in humans and could induce psychoactive effects within seconds and last for minutes in a dose dependent manner (30). It would be important to find whether an ideal dose of SA could be identified that has the neurological protective effect with minimal or no psychoactive effects. Brain HI has very narrow therapeutic time window, thus, it is critical in the future studies to investigate the optimal time point of SA administration and define therapeutic time window for SA after HI insult. All these should be determined in a model with definitive long term neurological impairment in the absence of intervention instead of using the current model.

Conclusion

In this study, the administration of SA is associated with improved neurological outcomes in several aspects and reduced mortality rate after hypoxic injury, suggesting that SA could be potentially served as an alternative treatment strategy for neonatal hypoxia.

Compliance with Ethics Requirements

All authors declare no conflict of interest.

All institutional and national guidelines for the care and use of laboratory animals were followed.

Acknowledgement: The authors would like to thank the support from Dr. Qingcheng Meng at the Department of Anesthesiology and Critical Care at the University of Pennsylvania, Perelman School of Medicine and the support from the Neurobehavior Testing Core at the University of Pennsylvania, Perelman School of Medicine.

Citation: Chunhua Chen1,*Xu Cui1,* Felipe Matsunaga1, Jingyuan Ma, Nan Ma, Ted Abel, Renyu Liu. Salvinorin A decreases mortality and improves neurological outcome in a neonatal mouse hypoxia model. Transl Perioper Pain Med 2014, 1(2):*****

Publication date:September 8^th, 2014