Protective effect of dexmedetomidine on reduction of endothelial glycocalyx shedding in patients undergoing surgery for traumatic brain injury-a randomized control trial.

Traumatic brain injury in the present day scenario is a major public health problem resulting in longterm disability and death  especially in young adults  The glycocalyx is a proteoglycan polymer  mainly consisting of proteoglycan and glycosaminoglycan chains  which is synthesized and secreted by endothelial cells and lies on the surface of the vascular endothelium  The most prevalent component of proteoglycans is syndecan 1 and the most prevalent components of glycosaminoglycans are heparan sulfate and hyaluronan  An intact glycocalyx is important in the determination of vascular permeability  mediation of nitric oxide release by shear stress sensing  and regulation of leucocyte adhesion and coagulation pathways  The structure of glycocalyx is extremely fragile and easily damaged by several factors  Increased oxidative stress causes the deterioration of the glycocalyx partly through the activation of heparinase  Post trauma elevations in syndecan 1 levels have been associated with coagulopathy and mortality  suggesting that degradation of the endothelial glycocalyx may contribute to the development of Acute trauma induced coagulopathy  The anesthetic agents can provide neuroprotection by maintaining an adequate balance between cerebral oxygen demand and supply  The protective effect of sevoflurane on the glycocalyx has been demonstrated in animal studies  In an isolated guinea pig heart study  electron microscopy demonstrated that sevoflurane treated hearts had an intact glycocalyx and sevoflurane treatment reduced the adhesion of platelets to the vascular endothelium  Sevoflurane has also been reported to protect the endothelial glycocalyx against ischemia reperfusion induced degradation   Syndecan 1 and HS will be also preserved by sevoflurane  sustaining the vascular barrier against ischaemic damage  However  its relevance in a clinical setting remains unknown  Dexmedetomidine is a sedative that acts on the  alpha2 adrenaline receptor and is commonly used in intensive care  DEX has shown promising results in reducing glycocalyx damage in animal models  Previous studies  such as Kobayashi et al   have demonstrated a reduction in serum syndecan 1 level and improvement in survival in animal models of haemorrhagic shock  but no human clinical trial has specifically assessed its impact on glycocalyx preservation in TBI patients

We therefore  hypothesize that dexmedetomidine based anesthesia would confer additional protection against TBI induced glycocalyx damage in clinical settings  If dexmedetomidine based anesthesia significantly reduces syndecan 1 and HS levels  this would offer strong evidence of its protective influence on the endothelial glycocalyx and introduce a novel approach for managing TBI and coagulation

AIMS and OBJECTIVE

Primary objective  To evaluate the syndecan 1 and heparan sulfate levels with dexmedetomidine based anesthesia in adult TBI patients undergoing surgery

Secondary objective  To evaluate the duration of mechanical ventilation and hospital length of stay  and neurological outcomes at 30 and 90 day post discharge extended Glasgow Coma Scale GOSE

Materials and Methods  Following approval of the Institute Ethics Committee  116 patients between 18 to 60 years of age  with moderate to severe TBI posted for emergency decompressive craniotomy at JPNATC  AIIMS  New Delhi will be enrolled for this study  A written  informed consent will be obtained from the patient or legally authorized representative prior to the study

Study design  A prospective randomized double blinded study

Inclusion criteria  Patients aged between 18 and 60  of either gender  belonging to ASA physical status I or II undergoing emergency decompressive craniotomy for traumatic brain injury requiring general anesthesia

Exclusion criteria  The exclusion criteria will be as follows

Patients with mild moderate head injury GCS more than 8

Patients with SBP less than 90 mmHg

Previous  history of heart disease or surgery

Hemodynamic instability requiring medical or mechanical attention

Severe hepatic and renal insufficiency  and

Severe chronic obstructive pulmonary disease

Randomization and Study Groups

A computer generated random number table will be used  and that will be concealed in an opaque sealed envelope until the start of anesthesia  When patients enter the operating room  the anaesthesiologist will open the opaque envelope and determine the anesthetic protocol

All the patients will be divided equally into two groups A and B

Group A Intervention group will receive sevoflurane and Dexmedetomidine Loading  1 microgram per kg and 05 microgram per kg per hr infusion for maintenance

Group B  Control group will receive sevoflurane with saline 09 percent infusion as placebo control   for the maintenance of anesthesia

The patient and the anesthesiologists will be blinded to the trial group assignments

Anesthetic technique  Each patient will undergo a pre anesthetic check up prior to shifting the patient inside the operation theatre  All patients enrolled in the study will receive general anesthesia with tracheal intubation with standard institutional protocol  On the day of surgery  the patients will be premedicated with glycopyrrolate 02 mg intramuscularly 30 min before the induction of anesthesia  In the operating theatre  monitoring modalities will include heart rate  non invasive blood pressure electrocardiography  pulse oximetry  end tidal carbon dioxide  and invasive blood pressure using 20 G intra arterial catheter in the radial artery  The patients will be positioned supine with the head and neck supported on pillows so that they will be as close to the neutral position as possible within their comfort range  Anesthesia will be induced with Propofol 1 to 2 mg per kg and fentanyl 2 microgram per kg rocuronium 1 mg per kg will be given to facilitate endotracheal intubation   After induction  in group A  anesthesia will be maintained with fentanyl 1 microgram per kg per h  rocuronium 01 mg per kg per h  sevoflurane 08 1 MAC  and Dexmedetomedine titrated to a BIS between 40 and 60  Whereas  in group B  anesthesia will be maintained with fentanyl 1  microgram per kg per h  rocuronium 01 mg per kg per h  sevoflurane  and 09 percent saline titrated to BIS between 40 and 60  The hemodynamic parameters heart rate  SBP  diastolic blood pressure  and mean arterial pressure will be recorded prior to induction of anesthesia as baseline values  intraoperatively at every 10 min interval till the end of surgery and 12 h after completion of surgery  The MAP will be kept above 65 mm of Hg  In case of hypotension  crystalloid fluid bolus of 3 to 5 ml per kg will be given initially followed by boluses of intravenous ephedrine 3 mg or phenylephrine 50 to100 microgram  If MAP persisted at less than 65 mm of Hg for more than 5 min  an infusion of noradrenaline will be started at the rate of 005 to 01 micro g per kg per min  In the case of hypertension  intravenous 2 to 3 esmolol boluses 03 to 05 mg per kg will be administered  In case of any bradycardia of less than 50 per min  intravenous atropine 05 mg will be administered  All rescue drugs used to maintain hemodynamics will be recorded and the urinary bladder will be catheterized in all the patients to monitor intraoperative urine output  The surgery will be conducted by an experienced neurosurgeon more than 2 years of experience in neurosurgery who will be blinded to the agent used for the maintenance of anesthesia  Fentanyl infusion will be stopped at the beginning of skin closure whereas the maintenance agents will be stopped following completion of skin closure  None of the patients will be extubated at the end of surgery and will be shifted to the neurosurgical intensive care unit  Fentanyl infusion will be stopped at the beginning of skin closure whereas the maintenance agents will be stopped following completion of skin closure  None of the patients will be extubated at the end of surgery and will be shifted to the neurosurgical intensive care unit

Data collection and Outcome measures

Pre operative data collected included sex  age  height  weight  BMI  blood pressure  blood glucose  and type of operation  Approximately 5 ml of radial arterial blood will be collected to analyze the concentrations of syndecan 1  and Heparan sulfate

Primary outcome  Syndecan 1 and Heparan sulfate levels with dexmedetomidine based anesthesia in TBI patients at various time points T0  T1  T3  andT4

Preoperative period just before induction of anesthesia to baseline T0

at the end of the surgery skin closure T1

6 hr after surgery T2 and

24 hr postoperatively T3

Secondary outcome  Duration of mechanical ventilation and hospital length of stay  and neurological outcomes at 30 and 90 day post discharge will be assessed using the extended Glasgow Coma Scale GOSE  Outcome will be dichotomized into favourable GOSE 5 to 8 and unfavorable GOSE 1 to 4 outcomes

Biochemical analysis

Sample Collection  Approximately 5 ml of radial arterial blood will be collected to analyze the concentrations of syndecan 1 and Heparan sulfate at the following time points  preoperative period just before induction of anesthesia T0  at the end of the surgery i e  skin closure T1  6 hr after surgery T2 and 24 hr postoperatively T3  Blood samples will be collected under sterile conditions using a heparinized arterial blood gas syringe to prevent coagulation

Sample Processing  The collected blood will be then immediately transferred into pre labelled anti coagulated tubes  The tube will be gently inverted four to five times to ensure proper mixing  The samples will be promptly transported on wet ice to the processing laboratory within 30 minutes of collection to minimize degradation of syndecan 1 and heparan sulfate  The blood samples will then be centrifuged for 4 degree C for 15 minutes at 3000 rpm to separate the plasma  The plasma will then be carefully extracted using a micropipette and then aliquoted into three per sample in a pre labelled cryovial

Storage  The aliquots will then immediately be stored at  80 degree C until batch analysis to maintain biomarker integrity and prevent freeze thaw degradation  Sample handling will be strictly monitored

ELISA test for Syndecan 1 and Heparan Sulfate  Serum levels of Syndecan 1 and Heparan sulfate will be measured by sandwich enzyme linked immunosorbent assay using commercially available kits with well defined specificity for syndecan 1 and heparan sulfate

Plate Preparation  A 96 well microplate pre coated with monoclonal antibodies specific for syndecan 1 and heparan sulfate will be used

Standard and Sample Loading  Standards of known antigen concentrations with test samples will be added to designated wells in duplicate to ensure accuracy  The plate will then be incubated for 120 minutes at room temperature

First washing  The wells will be emptied by inverting the plate on an absorbent paper towel  The plate will then be washed three times using the provided wash buffer  ensuring no liquid remains after each wash

Biotinylated Antibody Incubation  A biotinylated detection antibody will be added  followed by 90 minutes of incubation at room temperature

Second Washing  The plate will again be washed three times using the provided wash buffer  ensuring no liquid remains after each wash

Avidin Biotin Peroxidase Complex Incubation  The wells will be incubated with the Avidin Biotin Peroxidase complex for 40 minutes at room temperature

Third Washing  The plate will be washed five times to remove unbound enzyme conjugate

Colour Development  A colour developing reagent will be added  and the plate will then be incubated at room temperature in the dark for 30 minutes

Reaction Termination  A stop solution will be added in each well to halt the reaction

Absorbance Measurement  The optical density of each well will be measured at 450 nm using an ELISA plate reader

Quantification The density of the colour produced is proportional to the amount of test antigen present in the sample  The mean absorbance obtained from each set of duplicated standards and test samples will be subtracted from the blank optical density value  A standard curve with known concentrations of Syndecan 1 and Heparan sulfate antigen on the x axis and absorbance on the y axis will be plotted to calculate the concentrations of the test antigen using the straight line form of the equation

Sample size calculation  The sample size of a total of 116 will be calculated based on a previous study by Kobayashi et al taking an alpha error of 005 percentage at 80 percentage power  The study conducted by Kobayashi et al  showed that the mean difference in syndecan 1 between the two groups will be 02  the standard deviation of intra individual variation will be 04  and the effect size is 055  The calculated sample size using G Power software is 104 accounting for a 10 percentage dropout rate  the adjusted sample size is 116

Statistical analysis  Categorical data will be expressed as a number of patients and compared using the Pearson chi square test or Fischer exact test  Numerical data will be expressed as mean plus SD and compared using an independent student t test or Mann to Whitney U test  Shapiro Wilk test will be performed to test the normality of continuous data  All analysis would done at 005 level of significance  A 2 tailed value of p less than 005 will be considered statistically significant  Statistical analysis will be performed with STATA software