Cytoprotective effect and clinical outcome of perioperative progesterone in brain tumors. A novel randomized mono-centeric microscopic evidence based research.

Objectives. The primary end-point of the current study was to to provide contemporary estimate about both cytoplasmic and nuclear effects of intramuscular progesterone therapy prior to craniotomy. Secondary end-points were to track post-operative course and short term (3 months) neurological sequele. Cellular neuronal microscopic examination by immuno-histochemistry (Progesterone receptor density ) and by H&E. Allred score for nuclear staining for PR receptors . Post-operative course included time to wean from mechanical ventilation (hours), Length of ICU stay (days) and brain neuro-imaging by brain CT). Short term outcome included sensory, motor and autonomic assessment. difference between basal values, CT2 and CT3 values inside Control group (P values =0.07 and 0.08). Three cases (2.5%) only declared to have mild oedema around tumor margin and 6 (5%) cases with moderate brain oedema. Control group brain imaging revealed 11 case (8.7%) with mild peri-lesional brain oedema, 19 cases (15.2%) with moderate diffuse brain oedema, normal brain CT imaging declared in 96 cases (76 .1%).


Introduction
Progesterone is a naturally occuring neurosteroid that can offer neuroprotective effects via pleiotropic pathways. Progesterone decreases cerebral oedema (1) restores blood-brain barrier integrity (2), reduces the in ammatory response and prevents cellular necrosis and apoptosis. The compression of brain tissue by a tumour mass is believed to be a major cause of the clinical symptoms seen in patients with brain cancer. However, the biological consequences of these physical stresses on brain tissue are unknown. Here, via imaging studies in patients and by using mouse models of human brain tumours, we show that a subgroup of primary and metastatic brain tumours, classi ed as nodular on the basis of their growth pattern, exert solid stress on the surrounding brain tissue, causing a decrease in local vascular perfusion as well as neuronal death and impaired function. We demonstrate a causal link between solid stress and neurological dysfunction by applying and removing cerebral compression, which respectively mimic the mechanics of tumour growth and of surgical resection. We also show that, in mice, treatment with lithium reduces solid-stress-induced neuronal death and improves motor coordination. Our ndings indicate that brain-tumour-generated solid stress impairs neurological function in patients, and that lithium as a therapeutic intervention could counter these effects.
Brain tissue can be deformed and its function perturbed by two mechanical forces: tumour-associated edema and solid stress . Solid stress refers to the compressive and tensile mechanical forces exerted by the solid components of the tissues, such as cells and extra-cellular matrix The tumour growth-induced deformation of the brain -the so-called "mass effect" . (3) Primary outcome was to examine cytoplasmic and nuclear actions of intra-musculer progesterone amid surgical neuronal injury and tumor induced neuro-excitotoxicity. Secondery outcome was to explore short term outcome 3 months after craniotomy regarding sensory , motor and autonomic function.

Material And Methods
This prospective randomized double-blind mono-center study approved by the local ethics committee of the Faculty of Medicine, Minia University hospital under institutional review board number (623-4/2020), and registered on clinical trial (NCT04414020), involved 252 adult patients of both sexes, ASA I-II, who underwent elective craniotomy. Exclusion criteria included candidate refusal to participate in the study, emergency craniotomy, any risk for developing venous thromb-embolism (previous deep venous thrombosis, systemic lupus or cerebrovasculer stroke), tumor recurrence, population with stented coronary arteries, morbid obesity and giving relevant history of ovarian or endometrial neoplasia.
An informed written consent obtained from all participants who were randomly and equally allocated into two groups through web-based randomizer (https://www.randomizer.org). Computerized randomization was performed using variable block sizes. Opaque sealed envelopes were used for allocation concealment. Boxes with patient numbers containing unlabeled coded vials were provided for all patients. Saline prepared for injection was in equi-volume with progesterone. Serum progesterone was checked twice, rst immediately before initiating rst dose and the second one was taken at one week after therapy. Double blind fashion disputed (Patient allocation was concealed from patient and the histopathologist). Data acquisition was tasked by two anesthetic lecturers who were not informed about study design. Candidates were assigned in two equal groups, Control group received intramuscular saline (2ml-NaCL0.9%) as a placebo for ve days daily before and after surgery, while progesterone group (PR group) received intramuscular progesterone (1mg/ kg) for ve days daily before and after craniotomy.
Saline prepared for injection was in equi-volume with progesterone.
Both injectate, progesterone and placebo are similar regarding color, duration of therapy and route of injection. Serum progesterone was checked twice, rst immediately before initiating rst dose and the second one was taken at surgery morning. In both groups, general anesthesia and endotracheal intubation was accomplished with Propofol (2mg/kg) and Fentanyl (2 mcg/kg) and neuromuscular blockade by cis-atracurium (0.5 mg/kg). Anesthesia was maintained with sevo urane (2-4 %). Intraoperative monitoring included 5 lead elecrtocardiogram, invasive blood pressure, temperature probe, oxygen saturation, exhaled CO 2 (end-tidal capnography), train of four and bispectral index. Tube of polyvinyl chloride size 7 secured at the angle of the mouth with a stabilizer. Fixation decided to be on mouth angle opposite to surgical site. Full sensory and motor neurological examination were executed on 3 months visit post-operative.

2.1.Surgical biopsy
Tumors were excised following standard surgical techniques regarding positioning, skin incision, craniotomy, dural opening, dissection, hemostasis and closure. Biopsy obtained from multiple points at the tumor-brain interface by cotton tipped swab under cover of surgical microscope.

Case selection and tissue sample preparation
Para n blocks with clinico-pathological data of the patients were collected. H&E slides were prepared to detect histopathological changes as congestion, cytoplsmic edema, necrosis and in ammation.

Immunohistochemistry
Serial sections were cut 4μm thick on positively charged slides. The slides were de-para nized with xylene, rehydrated through graded ethyl alcohol. Then, they were immersed in 3% hydrogen peroxide for 30 min and rinsed in phosphate buffer solution (PBS). Citrate buffer (pH 6.0) was used for antigen retrieval by the microwave for 10 minutes. Sections were left to cool at room temperature then washed in PBS. Afterwards, rabbit polyclonal anti-PR anti-body (ready to use, Abcam) was incubated overnight at 4°c in humidity chamber. Slides were then rinsed with PBS before applying secondary antibody for 30 min. After wash in PBS, streptavidin-biotin complex was added for another30 min. Brownish color developed by using diamino-benzoate (DAB), then the slides were washed in distilled water. Lastly, they were stained with hematoxylin, dehydrated, cleared by xylene and covered slipped.

Evaluation of immunostaining.
According to Allred scoring system, The intensity score (IS) of nuclear staining evaluated as follows: 0= no positive cells, 1= mild positivity, 2= moderate positivity and 3= strong positivity. The proportion of staining power evaluated according to the percentage of positive cells: 0= negative, 1=1%, 2=2-35%, 3=36-65% and 4=66-100%. PR immune-expression score was calculated as the algebric summation of the scores for the intensity of staining and the extent of staining power. Final scores were then classi ed into low (< 3) and high (≥ 3) (0).

2.3.1.Sample Size Calculation:
Before the study runover, the number of patients required in each group was determined after a power calculation according to data obtained by a Pilot study performed on ten consented candidates, ve in each group. In that study, the percentage of cellular damage in Progesterone group was 20% ( one case with cytoplasmic oedem), while in Placebo control group was 80%( four cases with cytoplasmic degeneration). A sample size of 126 patients in each group was determined to provide 99% power for Fisher's exact test at the level of 0.05 signi cance using G Power 3.19.2 software. Statistical analysis -The collected data were coded, tabulated, and statistically analyzed using SPSS program (Statistical Package for Social Sciences) software version 20.
-Descriptive statistics were done for Parametric quantitative data by mean, standard deviation and minimum & maximum of the range, while they were done for categorical data by number and percentage.
-Analyses within each group were done for parametric quantitative data using paired sample t test, and for qualitative data using Wilcoxon signed rank test. Analyses were done for qualitative data using Fisher Exact test.
-The level of signi cance was taken at (P value < 0.05).
Results    The slide shows ruptured blood vessels, mild hydrophilic degeneration. Brain imaging 3 days postcraniotomy revealed moderate brain oedema, Three month follow up was free.

Discussion
Neuro-critical care residents questioned for a safe effective management for trauma or surgical induced neuronal injury. Biopsy based microscopic and immuno-histochemistry examination is a leap unique method that doesnot give chance for bias. Extensive literatures had led to a debate about the clinical e cacy of progesterone in providing neuro-quiescence and neuronal repair depending only on clinical course and neurological outcome (4). Novelty of this paper came from its use of neuronal biopsy, a new senior modality to deny or con rm cellular injury.
Despite the multi-factorial bene ts of progesterone obtained in the experimental models of TBI and the promising results of two Phase II clinical trials [5,6], two Phase III clinical trials failed to show bene ts of progesterone [7,8] . Among the concerns that have been raised were the diversity of the enrolled patients concerning sex, age, and severity of TBI and the different doses regimen of progesterone used.
Many recent nested randomized clinical trials reported that progesterone can provide cerebroprotection. Zhao etal 2019 (9) , in a wide spectrum meta-analysis consisting of eight randomized clinical trial hosted 2251 patients were xtured for both safety and e cacy of injected progesterone on TIBs population.
The regimen included 1 mg/kg intramuscular progesterone every 12 hours for ve days. Their results are compliant with us regarding candidates with progesterone group had less ICU time, better neurologic outcomes (RR =1.51; , P=0.007) than those who received placebo. Progesterone offered neuroprotection till 3 months after impact.
Neuronal injury in operable brain tumors is coming from two ways, rst is the direct mechanical compression by the tumor itself impeding adjacent healthy tissue blood supply and the other is surgical craniotomy. Surgical craniotomy induced cerebral injury is mostly due to direct parenchymal injury resulting from focal exertion of pressure on retracted neural tissue associated with the use of plate and self-sustaining retractors to access deep intra-parenchymal lesions. New methods were in use to minimize tissue injury including neuro-navigation and endoscopic approaches, but yet no full protection was declared . This was the point of interest dealt by Stephen etal., 2020(10) who linked post-craniotomy morbidity and mortality related to retractors induced ischemic injury. Patho-physiological mechanisms that can provoke neuronal injury following surgical head trauma is post-traumatic in ammation, which has been shown to increase blood-brain barrier (BBB) permeability and cerebral edema. During posttraumatic in ammation, metabolic products of arachidonic acid, known as prostanoids, including prostaglandins, prostacyclin and thromboxanes, are released. Following the overwhelming production of in ammatory mediators within the injured brain is believed to contribute to the cerebral damage and BBB dysfunction (11). Key mechanism in ameliorating surgery induced cerebral injury is combined action of progesterone and its active metabolite allo-pregnanolone

Results of preclinical analyses demonstrated that allo-pregnanolone is a potent inducer of neural progenitor proliferation of progenitor cells in vitro.
Liere etal., 2014 (12) in their paper about revisting the role of progesterone metabolite in central nervous system. Allopregnanlone signi cantly increased neurogenesis within the subgranular zone of the dentate gyrus and subventricular zone, maintaining selective controlled BBB permeability, promoted survival of young neurons and functional recovery(12). This was clearly identi ed in PR group biopsy slides and brain CT. Biopsy showed impedence to neutrophilic in ltration and blood vessel congestion. Also brain CT was demonstrative in PR group regarding decrease in perilesional oedema( cytotoxic brain oedema surrounding tumor). This action is explained by progesterone inhibitory action on in ammatory vasodilator substances (Nitric oxide and Interleukins) and by increasing endothelial progenitor cells (13) Dose choice was based upon a previous randomized controlled trials that used progesterone in traumtic brain injury in a dose 1mg/kg intramusculer for 5 days post-traumatic targeting length of ICU stay as a primary endpoint. (Hassan etal., 2017 ).
The current research endorsed enhanced transcription and upregulation of nuclear Progesterone receptors with enhanced uptake of staining ( both intensity and proportion are increase) in PR group. Candidates with these ndings showed smooth postoperative course ( less time for weaning and ICU stay). Both progesterone can suppress in ammatory mediators productions together with the gamma amino butyric acid (GABA) like action of its active metabolite allopreganolone which can bind to GABAa receptors mediating neuronal hyperpolrization via opening of inhibitory chloride channel. This GABA mimetic action increase neuronal level of inhibitory neurotransmitter glycine that counteract excitatory effects of glutamate that increase neuronal injury induced by tumor and craniotomy (15,16).
Our results run hand by hand with Hassan etal.,2017 (17) who injected progesterone intramuscularly in traumatic brain injury victims for ve days declaring less time for ICU stay and early weaning from mechanichal ventilation. Limitations.
2. Not all cases cooperated in biopsy harvesting .
3. Surgical hazards from biopsy obtaining. Availability of data and materials ALL data supporting results ( biopsy ) in the current research sent with submission for clarity. CT images sent upon request.

Competing interests
No competing interest Funding No speci c source of funding Authors' information All authors whose names appear on the submission 1) Made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data; or the creation of new software used in the work; 2) Drafted the work or revised it critically for important intellectual content; 3) Agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. 0.0001* *Wilcoxon signed-rank test for the intragroup data (against . baseline CT ), **Mann-Whitney U-test (change between groups). **P<0.05 is considered significant for intragroup comparison *P<0.05 is considered significant for comparison between groups. *Significance difference inbetween two studied groups( p value < 0.05) ** Significance difference between basal and other CT readings in studied groups( p value < 0.05) CT1= Brain CT accomplished before therapy initiation ( on admission ) CT2= Brain CT accomplished one week after therapy ( on surgery morning) CT3= Brain CT accomplished 3 days postoperative.

Tables
Extubation time= Time elapsed from transfer from operative theatre to neuro-ICU till weaning from assissted ventilation to spontaneous ventilation.    The slide shows ruptured blood vessels, mild hydrophilic degeneration. Brain imaging 3 days postcraniotomy revealed moderate brain oedema, Three month follow up was free.

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