Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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Dr Bhanu K Bhakhri

"The Journal of Clinical and Diagnostic Research (JCDR) has been in operation since almost a decade. It has contributed a huge number of peer reviewed articles, across a spectrum of medical disciplines, to the medical literature.
Its wide based indexing and open access publications attracts many authors as well as readers
For authors, the manuscripts can be uploaded online through an easily navigable portal, on other hand, reviewers appreciate the systematic handling of all manuscripts. The way JCDR has emerged as an effective medium for publishing wide array of observations in Indian context, I wish the editorial team success in their endeavour"



Dr Bhanu K Bhakhri
Faculty, Pediatric Medicine
Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
On Sep 2018




Dr Mohan Z Mani

"Thank you very much for having published my article in record time.I would like to compliment you and your entire staff for your promptness, courtesy, and willingness to be customer friendly, which is quite unusual.I was given your reference by a colleague in pathology,and was able to directly phone your editorial office for clarifications.I would particularly like to thank the publication managers and the Assistant Editor who were following up my article. I would also like to thank you for adjusting the money I paid initially into payment for my modified article,and refunding the balance.
I wish all success to your journal and look forward to sending you any suitable similar article in future"



Dr Mohan Z Mani,
Professor & Head,
Department of Dematolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
Knowledge is treasure of a wise man. The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."



Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help ones reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journalsNo manuscriptsNo authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011

Important Notice

Experimental Research
Year : 2010 | Month : October | Volume : 4 | Issue : 5 | Page : 3304 - 3311

Beneficial Effects Of Inhaled Nitric Oxide On Lung Pathology And Energy Metabolism In A Canine Model Of Smoke Inhalation Injury

SHUNZHEN QI*1, MENGJUN WANG†1, WEIHAI SUN§, VALERIO ZACÀ‡, WEN LI°, RONG XIU#, AND HONGLIANG ZHAO¶ Correspondence Address :
Mengjun Wang, MD, Cardiovascular Research, Henry Ford Health System, 2799 W Grand Blvd., Detroit, MI 48202, USA. Phone 1-313-916-4023; Fax 1-313-916-3001; E-mail: mwang1@hfhs.org.

Abstract

Nitric oxide (NO) exhibits positive effects in the treatment of acute lung injury (ALI); angiotensin converting enzyme (ACE) has been indicated as a marker of pulmonary endothelium damage in ALI. We examined the effects of inhaled NO on ACE activity, pulmonary pathology and energy metabolism in a canine model of smoke inhalation injury. Following smoke exposure, 17 dogs were randomly assigned to receive a mixture of NO (45 ppm) and O2 (FiO2=45%) (Treatment group, n=9) or O2 alone (FiO2=45%) (Control group, n=8) for 12 hours. As compared to O2 alone, NO therapy effectively limited the increase in ACE activity and preserved lung ATP level and energy charge. Moreover, NO exerted a protective effect on the extensivemorpho-structural changes which were observed in the dogs who received O2 alone. The present study demonstrates that inhaled NO after smoke inhalation injury may exert beneficial effects that are likely to be due in part to a protective effect on pulmonary endothelium and pulmonary haemodynamics.

Keywords

Nitric oxide, acute lung injury, pulmonary endothelium, angiotensin converting enzyme, inhalation therapy, ATP

INTRODUCTION
Pulmonary vascular endothelial damage is a common pathological feature in the early phase of most forms of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) (1)-(4). This damage results in the loss of endothelium-derived vasodilators, with diffuse vasoconstriction and increase in pulmonary vascular resistance, as well as in the microvascular hyperpermeability to both fluid and proteins, with consequent interstitial and intra-alveolar oedema (1)-(4). Lung endothelial cells are the major sites of the production of the angiotensin converting enzyme (ACE) (5) and several studies have suggested a role for the evaluation of plasma and lung ACE activity as a marker of pulmonary damage, both in experimental and clinical settings of inhalation injury (6), (7). In the recent years, inhaled nitric oxide (NO) therapy has been extensively used to treat a wide variety of lung diseases (as reviewed in reference 8). Acting as a selective pulmonary vasodilator, inhaled NO has been demonstrated to improve ventilation-perfusion matching, thus ameliorating oxygenation and lowering pulmonary vascular resistance in patients suffering from ALI and ARDS (8), but it is not clear what the effects of inhaled NO on pulmonary ACE activity, energy metabolism and pathology are. Previous studies from our laboratory and others showed that inhaled NO may exert beneficial effects in animal models of smoke inhalation injury, thus improving oxygenation, pulmonary hypertension and microvascular resistance (9)-(12). The objective of this study was to examine the effects of inhaled NO on plasma and lung ACE activity, energy metabolism and pathology, by using a canine model of smoke inhalation injury. We hypothesized that inhaled NO would decrease plasma and lung ACE activity, preserve pulmonary energy metabolism and reduce lung histological damages.

Material and Methods

Animal model
The canine model of smoke inhalation injury which was used in the present study was previously described in detail (9), (13). In summary, 17 healthy age- and weight-matched male cross-bred dogs were surgically instrumented under general anaesthesia and sterile conditions. Anaesthesia was induced with the intravenous administration of 3% sodium pentobarbital (30 mg/kg) and was maintained with a continuous infusion of barbiturate in order for it to be effective during the whole procedure. A 9F tracheal tube was introduced through a tracheotomy, a 7F three-channel Swan-Ganz catheter was advanced into the pulmonary artery via the right femoral vein and a 4F multipurpose catheter was advanced into the abdominal aorta via the right femoral artery for blood-pressure monitoring and arterial blood sample collection. Inhalation injury was induced by introducing a dense smoke which was produced by burning 150 g of white pine wood sawdust, into the dogs’ respiratory tract through the tracheal tube. The noxious chemicals and gases which were generated from the incomplete combustion of sawdust are formaldehyde, acetaldehyde, methane, formic acid and carbon monoxide. The respiratory rates and volumes were controlled by using a ventilator and they consisted of 18 breaths per minute of 320-350 ml (adjusted per dog’s weight) of smoke in each breath. The dogs underwent 4 minutes of continuous smoke inhalation, followed by 5 minutes of room air ventilation and an additional 90 seconds of smoke inhalation. From our experience, we found that such exposition to smoke is sufficient to cause a severe inhalation injury. The dogs were then ventilated (Newport, E-200 ventilator) after receiving an intravenous succinylcholine loading dose of 1.5 mg/kg, followed by continuous infusion (30 drops/h), with a tidal volume of 13 ml/kg, respiratory rate of 18 breaths/min, an inspiration/expiration ratio of 1:1.5 and a constant PCO2 level of 30 mmHg (with the aid of a CO2 sensor – CO2 monitor HP 14361A). Thirty minutes after the last smoke inhalation, the dogs were randomly assigned to be ventilated with a mixture of NO (45 ppm) and O2 (FiO2=45%) (Treatment group, n=9) or with O2 alone (FiO2=45%) (Control group, n=8); the temperature of the inhaled gas did not exceed 40° C. The concentrations of NO and NO2 in the inspired gas were continuously monitored by using a gas analyzer (42C-NO-NO2-Nox, USA). Twelve hours after the last smoke inhalation, the dogs were euthanized and the lungs were rapidly removed for biochemical and histopathological examination. Assuming the severe inhalation injury to be equivalent to 30% of the total body surface area (TBSA) which was burned, during the experiment all the dogs were hydrated according to the Parckland formula (4 ml/kg/% TBSA burned/24h). The lung tissue samples were obtained from 4 non-injured, non-treated dogs (Sham group, n=4) for comparisons. The experimental protocol was approved by the Experiment Animal Committee of the Bethune International Peace Hospital. The animals were handled in adherence to the Guidelines of the Chinese Association for Physiological Sciences.

Measured variables
Hemodynamic measurements. The mean pulmonary artery pressure (mPAP) and aortic pressure (AoP) were recorded invasively at each study time-point.
Biochemical analysis. Arterial blood samples were collected at the baseline (prior to any smoke inhalation) and 0.5, 2, 5, 8 and 12 hours after the last smoke inhalation for the assessment of blood gases, carboxyhaemoglobin (HbCO) and nitrate content. Plasma was extracted as a supernatant after centrifugation of 2 ml of heparinized blood at 3000 rpm for 10 minutes and was then stored at a temperature of –20˚ C. In order to obtain bronchoalveolar lavage (BAL), the superior lobe of the left lung was irrigated with 30 ml of 0.9% saline solution via a principal bronchus, followed by aspiration of the liquid after 1 minute. This procedure was repeated 3 times with a total of 90 ml of saline solution; the overall amount of lavage liquid which was obtained, was then centrifuged at 1000 rpm for 5 minutes and the supernatant was collected and stored at –20˚ C. A sample of the left lung tissue, weighing approximately 150 mg, was homogenized in a solution of 0.9% NaCl at 4˚ C and was centrifuged at 3000 rpm for 20 minutes. The supernatant was then extracted and preserved at –20˚ C. ACE activity in plasma, BAL and lung tissue was determined by utilizing the synthetic ACE-specific substrate hippuryl histidyl leucine (HHL); ACE activity was quantified from the moles of hippuric acid (HA) formed, in time-fixed assays, by using high performance liquid chromatography (HPLC) separation of HA from HHL and UV-spectrophotometry for the quantitation of HA as in the standard Cushman and Cheung assay (14). Colloid osmotic pressure (COP) in plasma and BAL was assessed by using a colloid osmometer. The total protein content in BAL was determined after Coomassie blue staining with an ultraviolet spectrophotometer (model DU-7 Beckman) with a reading wave-length of 595 nm; cattle serum protein was used to draw a standard comparison curve. The energy metabolism of the pulmonary tissue was estimated by reverse phase HPLC (15). The actual content of ATP, ADP and AMP in the tissue samples was calculated as the ratio of the sample spike square and the standardized spike square (corresponding to 25 μg/ml concentration of ATP, ADP and AMP). Energy charge (EC) was calculated according to the formula:
EC = (1/2ADP + ATP)/(ATP + ADP + AMP).
Histopathology and morphometry. We measured the lung water content according to the lung wet-to-dry weight ratio (W/D). For this purpose, the excised right lung was first weighed in a dry plate (wet weight – W). It was then weighed again after being dried at 80˚ C for 72 hours (dry weight – D). The W/D ratio was calculated as follows: W/D % = [(W – D)/W] x 100. The diaphragmatic lobe of the left lung was processed for histopathological examination. The tissue samples were fixed in 10% formalin, were embedded with paraffin wax, sectioned and stained with Hematoxyline-Eosine (HE) for light microscopy, or were fixed in 2.5% buffered glutaraldehyde and prepared routinely for transmission electron microscopy (TEM).

Statistical analysis
We adopted ANOVA and the Student’s t test for comparisons as and when appropriate. The data are reported as mean ± SEM; a p value < 0.05 was considered to be statistically significant.

Results

All animals survived the observation period. NO inhalation had no effects on mAP, but had effects on mPAP which decreased after 2 hours. The dogs exhibited the usual modifications in gas exchanges and HbCO levels which were associated with smoke inhalation injury; arterial blood methaemoglobin concentration resulted in <2% during the whole experiment in both study groups (data not shown). As expected, NO inhalation translated into higher arterial blood nitrate concentrations at each study time-point [Table/Fig. 1]. ACE activity. Comparisons of plasma ACE activity between the study groups are shown in (Table/Fig 2). Early after injury, there was an increase in the ACE activity in the overall study population. In the Control group, the increase in ACE activity reached statistical significance after 5 hours (p<0.05), resulting in nearly doubled values after 12 hours as compared to the baseline values. The NO treatment induced a significant decrease in the plasma ACE activity as compared to the controls.ACE activities in BAL and lung tissue were dramatically increased in dogs who were exposed to inhalation injury as compared to the Sham dogs (p<0.01, (Table/Fig 3)). However, NO inhalation was effective in significantly reducing the ACE activity in BAL (20.13±0.23 Vs 32.46±0.39 nmol/mgprot/min, p<0.01) and lung tissue (2.16±0.25 Vs 3.96±0.42 nmol/mgprot/min, p<0.01) as compared to O2 inhalation alone. COP. Following inhalation injury, plasma COP was decreased in the Control group (significantly decreased after 8 hours, p<0.05) and was substantially unchanged in the Treatment group, which was suggestive of a preserved COP in dogs who were randomized to NO inhalation (Table/Fig 4). Smoke exposure was associated with an impressive increase in COP and total protein content in BAL (Table/Fig 5); NO inhalation exhibited a significant protective effect with regards to these variables as compared to O2 inhalation alone (total protein content: 5000±470 Vs 6290±450 g/L, p<0.01) (Table/Fig 5).Energy metabolism. The dogs who were exposed to smoke inhalation injury exhibited a severe impairment of lung tissue energy metabolism (Table/Fig 6). However, in the Treatment group, we observed a less severe depletion of ATP (11.53±1.03 Vs 6.79±0.78 μmol/mgprot, p<0.01), ADP (3.05±0.36 Vs 2.73±0.31 μmol/mgprot, p<0.05), and EC (0.77±0.04 Vs 0.67±0.06, p<0.01) than in the controls.Histopathology. The W/D ratio was significantly increased (p<0.05) in the dogs who were exposed to inhalation injury as compared to the Sham dogs (75.58±6.07%); however, no substantial difference was observed between the Control and the Treatment groups (W/D: 84.32±4.01 Vs 83.32±6.08%, p=NS). Light microscopic examination of the lung tissue samples from the Control dogs [Table/Fig. 7 A] revealed marked tissue haemorrhage and congestion, thickening of the alveolar wall with alveolar oedema and an intra-alveolar collection of inflammatory cells, deciduous epithelial cells and erythrocytes; some alveoli appeared to be ruptured and syncretized. Analysis of the samples from the Treatment group [Table/Fig. 7 B] revealed an apparently less severe degree of morpho-structural damage, because the damage was displayed with mild tissue haemorrhage and congestion, preserved alveolar anatomy and a modest infiltration of inflammatory cells and erythrocytes.TEM analysis showed interstitial oedema, derangement and the rupture of the fibrous structures, the casting of type II alveolar cells in the alveolar cavity with oedema and incrassation and the loss of integrity of the basal membrane in the samples which were collected from the dogs in the Control group [Table/Fig. 8 A]. The integrity of the alveolar-capillary membrane appeared instead to be maintained and the cellular damages appeared milder, with the conservation of the organization of the lamellar bodies in the lung tissue samples from the Treatment group [Table/Fig 8 B].

Discussion

In patients with ALI or ARDS, NO inhalation has been shown to improve oxygenation without affecting the outcome (8). However, evidence from experimental studies has suggested that NO which is given after inhalation injury, may exert several additional positive properties beyond the sole improvement of oxygenation. Ogura et al. reported that inhaled NO significantly attenuated pulmonary vasoconstriction and pulmonary arterial hypertension, thus moderately improving the ventilation-perfusion mismatch in an ovine model of smoke inhalation injury (10), (11). Recently, Enkhabaatar et al. showed that inhaled NO which was given 22 hours after injury, along with reducing pulmonary vascular resistance and pulmonary artery hypertension, was effective in reducing microvascular hyperpermeability and lung water content (12). No change in aortic pressure was found to be related with NO inhalation in these studies.

The results of the present study confirm the effects of inhaled NO on the improvement of oxygenation and pulmonary arterial hypertension which were observed in these previous studies; furthermore, our data provides the first evidence that NO inhalation therapy exhibits beneficial effects on lung pathology and energy metabolism, thus limiting the severe damages which are associated with smoke inhalation injury. These beneficial effects may in part be explained by a potential protective effect which is exerted by inhaled NO on pulmonary vascular endothelial cells (6), (7), as suggested by lower ACE activities which were observed in the treatment group. ACE is a non-specific dipeptidylcarboxy-peptidase that removes COOH-terminal dipeptides from a variety of substrates, including bradykinines and it also catalyzes the conversion of Angiotensin I into Angiotensin II (5). Although it is present in many tissues, this enzyme is mainly expressed on the plasma membrane of the pulmonary vascular endothelial cells (5). Numerous studies indicated a role for ACE activity as a specific index of pulmonary endothelial damage of different aetiologies (16)-(19). Specifically, a significant increase in ACE activity has been observed in plasma, BAL and lung tissue in patients and animals after smoke inhalation injury (6), (7). Our observations showed that plasma ACE activity was increased early after injury and the further increase which was detected in the course of the experiment, might have potentially paralleled progressive endothelial cell damage. Along with a significant reduction in the plasma levels, which was observed as compared to the controls, the protective effect of NO inhalation was suggested by a lower expression of ACE activity, both in lung tissue and BAL.

Pulmonary microvasculature hyperpermeability to fluid and protein is observed early after inhalation injury and it is a direct expression of endothelial cell damage (1)-(4). Endogenous tachykinines and hydroxyl radicals are likely to represent the mediators of increased permeability (20)-(22), which in turn, lead to the extravasation of plasma and macromolecules into the interstitial space, with perialveolar and submucosal peribronchial and peritracheal oedema and into the alveolar cavity through a loss of integrity of the alveolar-capillary barrier. In the present study, inhaled NO was effective in significantly reducing BAL COP and total protein content when compared to O2 therapy alone; these results appear to be likely to be achieved by limiting endothelial cell dysfunction, thus halting the tendency to increase the permeability.

Although we observed a positive effect of inhaled NO COP increase and protein accumulation, both in BAL and lung tissue, which was suggestive of limited vascular hyperpermeability, the lung W/D ratio appeared to be substantially unchanged in the control and the treatment groups, even if it was sensibly increased as compared to the non-injured sham dogs. A potential explanation for this apparent lack of effect of NO inhalation on the lung water balance might be the limitation of our observation period to 12 hours, which was intended by design to investigate the acute therapeutic effect of NO in the setting of smoke inhalation injury. An early increase (i.e. < 12 hours following smoke exposure) in extravascular lung water content is very uncommon in patients suffering from inhalation injury and it is a result of the direct chemical toxicity of inhaled gases (23)-(25). A significant increase in extravascular lung water is usually first seen in the first 24 hours, with persistently elevated values for more than 48 hours (23)-(25). However, the available data from the literature, which is relative to the effects of inhaled NO on the pulmonary water content in inhalation injury, it still appears to be controversial (26)-(28).

Microvascular damage with oedema and increased pulmonary vascular resistance, along with airway obstruction significantly impairs oxygenation and lung tissue blood supply, thus negatively affecting the cellular energy metabolism, ATP synthesis and survival (21), (29). Mammalian cells depend on ATP to maintain their cellular activities and structural integrity; ATP depletion translates into cellular dysfunctions and ultimately, death. Compared to the Sham group, there was a severe depletion of ATP, ADP and EC in the Control group. However, in the Treatment group, we observed a less severe depletion of ATP, ADP and EC than that which was seen in the controls. Dogs who were exposed to smoke inhalation injury exhibited a severe impairment of lung tissue energy metabolism. Inhaled NO preserved the lung tissue production of ATP, thus contributing to the maintenance of a normal EC, potentially through improvement in pulmonary haemodynamics and oxygenation (9)-(12).

The pathological correlates of smoke inhalation injury are acute cell membrane damage with oedema and hyperaemia of the tracheo-bronchial mucosa (which progressively leads to necrotic tracheo-bronchitis with pseudo-membranes formation and airways obstruction), interstitial and intra-alveolar congestion and oedema due to damages of the alveolar epithelial barrier and lung endothelium (30)-(32). In our study, we focused mostly on the latter aspects. The morphological changes occurring in the alveolar epithelium classically included intracellular oedema with focal bleb and vescicle formation in the type-I alveolar cells and alterations in the membrane-bound vacuoles of the type-II cells; endothelial cells showed similar changes, although they were less severe (30)-(32). This pattern of lesions was observed in dogs who were treated with O2 alone after smoke inhalation injury; rather, NO inhalation therapy resulted in a significant limitation of microscopic damages of the alveolar and the endothelial cells. We might thus hypothesize that this positive effect is obtained through NO-mediated improvement in oxygenation and pulmonary energy metabolism (33), (34). Further investigations are needed, however, to clarify as to which mechanisms underlie the mitigation of the histological damages which were observed in NO-treated dogs.

In conclusion, the present study demonstrates that inhaled NO, when administered early after smoke exposure, can significantly decrease plasma and lung ACE activity, preserve pulmonary energy metabolism and reduce lung histological damages in a canine model of inhalation injury. These beneficial effects are likely to be due, in part to a protective effect which is exerted by NO on the pulmonary vascular endothelial cells and pulmonary haemodynamics.

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