SUTURE VERSUS FIBRIN GLUE MICRONEURAL ANASTOMOSIS OF THE FEMORAL NERVE IN SPRAGUE DEWLY RAT MODEL. A COMPARATIVE EXPERIMENTAL ASSESSMENT OF THE CLINICAL, HISTOLOGICAL AND STATISTICAL FEATURES.

INTRODUCTION: Peripheral nerve injury is a frequently encountered clinical problem that leads to functional losses at the long-term. Although microsurgical repair has been introduced to clinical practice in peripheral nerve injuries, unsatisfactory outcomes regarding functional recovery in target organ cause an increasing interest on studies about nerve injury and biology of the recovery in nerve injuries1. MATERIAL AND METHODS: Sciatic nerves of seventy adult Sprague Dewly rats were transected and primary anastomosis was performed. Rats were then divided into three groups: Control group, while 30 rats were repaired with sutures, and the remaining 30 were repaired with fibrin glue. After 30 days the rats were sacrified and the sciatic nerves were investigated histologically with morphometrical and statistical analyses. RESULTS: In microsurgical nerve repair, suture placement has been thought to cause hindrance to the sprouting axons and compress the blood supply to the fascicles, thereby impairing the regeneration of the transected nerve ends after repair, with possible neuroma formation. On the other hand, fibrin glue is a simple, effective technique, less time consuming than suturing. Another advantage of this suture-free technique is that it avoids injuring the axon with needles, and the lack of foreign bodies minimizes the inflammatory reaction. CONCLUSION: We recommend using fibrin glue as it demonstrates less inflammatory reaction, less scar tissue formation, it is less time consuming and provides better outcomes.

Changes in blood flow in ovine follicles and serum concentration of estradiol 17 beta (E2) and nitric oxide (NO) around the time of ovulation in Ossimi ewes.

The aim of the present study was to examine the relation between follicular blood flow of the ovulatory follicle and the levels of serum E2 and nitric oxide (NO) in Ossimi ewe. Seven cyclic ewes were synchronized with a double injection PGF2α. The follicular wave was examined daily until ovulation (disappearance of the large dominant follicle ultrasonographically) with transrectal color Doppler ultrasonography (8-10MHz linear array transducer). The number of recruited follicles was 4.8±0.9 (3-8 follicles) with diameter of 2.8±0.1mm. The interval from PGF2α injection to follicle deviation was 2.35±0.07 days. The diameter of the first largest follicle (LF1) at recruitment day was 4±0.3mm while the diameter of the second largest follicle (LF2) was 3.7±0.1mm. The diameter of LF1 at the day of deviation was 5.1±0.5mm while the diameter of the LF2 was 4±0.7mm. The diameter of the ovulatory follicle was 6.1±0.5at day of ovulation. We detected the blood flow area of the ovulatory follicle at D2. At ovulation, the blood flow area and blood flow area percent increased significantly to be 11.9±0.6mm(2) and 44±3.4% respectively. The results showed a positive correlation between E2 and NO (r=0.85, P<0.009). Both increased concomitantly with the diameter of the ovulatory follicle. Besides, NO and E2 reached a maximum level at ovulation (12.1±1.8ng/ml and 16.4±1.7pg/ml respectively).

Effect of major burns on early and late activating markers of peripheral blood T lymphocytes.

It is known that lymphocytes immunophenotype is a reflection of the functional level of the immune system. The immunosuppressive effect of major burns is also known for many years. T lymphocytes of 50 major burn patients were analyzed in base line (BL) samples at 24 hours and at 1 week and 2 weeks after burn, using monoclonal antibodies of CD3, CD4, CD8, CD25 (IL2R) and HLA-DR by flow cytometry and ß2-microglobulin (ß2-m) by ELISA. Recorded values were compared with those of 50 healthy donors. There was statistically significant reduction in absolute number of CD3 positive cells (CD3+) (p<0.000) and CD4/CD8 ratio (p=0.01) in the first 24 hours in comparison with controls. CD25 (IL-2R) shows insignificant upregulation on T lymphocytes after burn with significant upregulation of HLA-DR. The absolute number of CD3+ cells began to increase after 2 weeks (p=0.03) but remained less than controls (p=0.08). CD4/CD8 ratio was more or less same as healthy controls after 2 weeks. Upregulation of CD25 was insignificantly increased and that of HLA-DR was markedly increased after 2 weeks (p=0.001). Significant negative correlations were detected between mean values of ß2-m and both absolute numbers of CD3 and CD4 positive cells in BL and one week samples. In addition there was significant correlation between mean values of ß2-m and values of CD25 expression in the BL samples. The obtained data is suggestive of persistent activation of T lymphocytes two weeks after major burns whereas early shedding of ß2-m is related to activation of lymphocytes increasing their susceptibility to apoptosis, both indicative of altered immune response. Burn intensivists and surgeons should be keen to support the patients' immune system in the first hours following major burns. This support will ensure free-bacteremic blood with a consequent better prognosis.

Protein profiling for cancer biomarker discovery using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and infrared imaging: a review.

Cancer biomarker refers to a substance or process that is indicative of the presence of cancer in the body. A biomarker might be either a molecule secreted by a tumor or it can be a specific response of the body to the presence of cancer. Cancer biomarker-based diagnostics have applications for establishing disease predisposition, early detection, cancer staging, therapy selection, identifying whether or not a cancer is metastatic, therapy monitoring, assessing prognosis, and advances in the adjuvant setting. Full adoption of cancer biomarkers in the clinic has to date been slow, and only a limited number of cancer biomarker products are currently in routine use. Among proteomic technologies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) is a technique that has allowed rapid progress in cancer biology. Different further developed methods including e.g. SELDI (surface-enhanced laser desorption/ionization) and MELDI (material-enhanced laser desorption/ionization) are simple and high-throughput techniques that analyze with high sensitivity and specificity intact proteins expressed in complex biological mixtures, such as serum, urine, and tissues. The combination of mass spectrometry (MS) with infrared (IR) spectroscopic imaging is an attempt to combine different technologies in systems analytics. Both MALDI-TOF and infrared tissue imaging enable studying proteins distribution in tissue samples with a resolution down to 50 and 5 µm, respectively. In this review, we summarize recent applications and the synergistic combination of these new technologies to proteomic profiling for cancer biomarker discovery.

Development and application of Fourier-transform infrared chemical imaging of tumour in human tissue.

Fourier-transform infrared (FT-IR) based mapping and imaging is a fast emerging technology which is being increasingly applied to investigate tissues in the high-throughput mode. The high resolution close to the cellular level, the possibility to determine the bio-distribution of molecules of interest (proteins, peptides, lipids, carbohydrates) without any pre-treatment and the offer to yield molecular structure information have brought evidence that this technique allows to gain new insights in cancer pathology. Thus, several individual mainly protein and peptide cancer markers ("biomarkers") can be identified from FT-IR tissue images, enabling accurate discrimination between healthy and tumour areas. Optimal data acquisition (spatial resolution, spectral resolution, signal to noise ratio), classification, and validation are necessary to establish practical protocols that can be translated to the qualitative and quantitative clinical routine analysis. Thereby, the development of modern fast infrared imaging systems has strongly supported its acceptance in clinical histopathology. In this review, the necessity of analysis based on global cancer statistics, instrumental setups and developments, experimental state of the art are summarised and applications to investigate different kinds of cancer (e.g., prostate, breast, cervical, colon, oral cavity) are shown and discussed in detail.

Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for micro-high performance liquid chromatography.

In the paper we demonstrate a new approach for the preparation and application of continuous silica bed columns that involve encapsulation (entrapment) of functionalized silica microparticles, which can be used as packing material in micro high performance liquid chromatography (micro-HPLC) and capillary electrochromatography (CEC). Like traditional packed columns, these capillaries possess characterized silica particles that offer high phase ratio and narrow pore size distribution leading to high retention and separation efficiency, respectively. More importantly, immobilization of the microparticles stabilizes the separation bed and eliminates the need for retaining frits. The developed capillary columns were fabricated in exactly the same way as a packed capillary column (slurry packing) but with an additional entrapment step. This immobilization of the packed bed was achieved by in situ polymerization of styrene and divinylbenzene in presence of decanol as a porogen and azobisisobutyronitrile as thermal initiator. Silica particles with different particle sizes and pore sizes ranging from 60 to 4000 A were studied. In addition different modified silica was used, including C-18 reversed phase, anion exchange and chiral stationary phases. Efficient separation of polyphenolic compounds, peptides, proteins and even DNA mutation were achieved using the developed technique depending on the properties of the silica particles used (particles pore size). For example, using 3 microm ProntoSIL C-18 particles with 300 A pore size, separation efficiencies in the range of 120,000-200,000 plates/m were obtained for protein separation, in a 6 cm x 200 microm i.d. capillary column. Using encapsulated silica C-18 with 1000 A pore size, separation of DNA homo and hetero duplexes were achieved under denaturing HPLC conditions for mutation detection. In addition, nucleotides were separated using anion exchange material encapsulated with poly(styrene-divinylbenzene) (PS/DVB), which indicated that the chromatographic properties of the silica packing material were still active after polymerization. The prepared capillary columns were found to be stable and could easily be operated continuously up to a pressure of 350 bar without column damage and capillary can be cut to any desired length.

Derivatized nanoparticle coated capillaries for purification and micro-extraction of proteins and peptides.

Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. One of the most powerful methods is affinity purification, also called affinity chromatography, whereby the proteins of interest are purified by virtue of their specific binding properties to an immobilized ligand. Affinity purification is becoming more widely used for exploring post-translation modifications and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. Our work was aimed to immobilize proteins or ligands for affinity purification of antibodies, fusion-tagged proteins and other proteins and peptides. Selected proteins or peptides are efficiently extracted and enriched using chemically derivatized walls of a fused silica capillary column. In this paper, we present an open tubular capillary, where the inner wall of a fused silica capillary was derivatized by covalent binding of modified polystyrene latex particles. The capillaries were derivatized with iminodiacetic acid and loaded with Fe3+ or Ni2+ for the purification and enrichment of phosphopeptides or His-tagged proteins, respectively. The latex coated capillaries have been successfully applied to enrich phosphopeptides from beta-casein tryptic digest and ovalbumin tryptic digest at a micro volume scale with recoveries ranging from 92 to 95%. The capillaries have been eluted under conditions compatible with MALDI-MS without any prior desalting step. In another approach, concanavalin A (Con A) or Protein G were immobilized on the epoxy modified latex on the inner wall of the fused silica capillary for the purification of glycoproteins and immunoglobulin, respectively. The design of the capillary and the protocols used for purification permits the direct detection of eluted proteins and peptides with gel electrophoresis or with mass spectrometry. The elution volumes are passed as discrete segments of few microliters over the inner surface of the open-tube capillary, achieving enrichment factors of more than 20-fold from starting samples.

Utilization of carbon disulphide for the analytical determination of betahistine hydrochloride and captopril in their pharmaceutical preparations.

Spectrophotometric, atomic absorption spectrometric and high performance liquid chromatographic (HPLC) procedures have been developed for the determination of betahistine hydrochloride and captopril. The three procedures are based on the reaction of the drugs with carbon disulphide in alkaline medium with the formation of the dithiocarbamate or the trithiocarbonate derivative of betahistine (BHT) and captopril (CAP), respectively, then subsequent chelation with divalent metals. The absorbance measurement of the formed chelates or of the metal moiety of chelates was used as the basis for the spectrophotometric and atomic absorption spectrometric determinations. The formed complexes have been used as pre-column derivatizing procedure for the HPLC determination of the two drugs. The different experimental conditions were optimized. The calibration graphs were linear over the applicable concentration ranges. The proposed procedures were applied successfully for the determination of the two investigated drugs in their tablets dosage form.

Spectrophotometric investigation of the formed chelate between timonacic and palladium(II) and its analytical applications.

A simple spectrophotometric method for the determination of timonacic is presented. The procedure is based on the chelate formation with palladium(II) chloride in buffered medium. The optimum conditions for the complex formation were ascertained and the method was developed for the determination of timonacic in the concentration range of 28-48 micrograms ml-1. The emperical formula of the formed complex was determined, by applying different spectrophotometric methods, at optimum pH of 4.8 and an ionic strength of mu = 0.5. The stoichiometric ratio was found to be 2:1 (timonacic/palladium) as calculated by the mole ratio, continuous variations and Asmus methods. The continuous variations and Nash methods were applied for the determination of the conditional stability constant of the formed yellow-water soluble complex and was found to be 3.27 x 10(7). The proposed methods was found to be suitable for the determination of timonacic in bulk and in its pharmaceutical tablets.

Stability-indicating assay of mequitazine in bulk and pharmaceutical tablets.

Simple and selective spectrophotometric procedures for the determination of mequitazine in its pharmaceutical tablets are described. The method was based on the formation of complex between mequitazine and palladium in presence of methyl cellulose in buffered or unbuffered media. The two procedures provide a mean of following the oxidative decomposition of the investigated drug. The apparent molar absorptivities at 460 and 500 nm were 2810 and 2840 and with Sandell's sensitivity of 0.116 and 0.103 for the buffered and unbuffered solutions, respectively. At the same time, Beer's law was obeyed in a concentration range of 2.4-7.2 mg% and the regression line equations were derived with correlation coefficients of 0.9997 and 0.9999 for buffered and unbuffered reactions, respectively. The validity of the method was further confirmed using the standard addition method. The proposed procedures demonstrate high percentage of recovery with good accuracy and precision.

Spectrophotometric and spectrofluorimetric estimation of ciprofloxacin and norfloxacin by ternary complex formation with eosin and palladium(II).

Spectrophotometric and spectrofluorimetric methods for the determination of two broad-spectrum fluoroquinolone antibacterials (ciprofloxacin and norfloxacin), either in pure form or in tablets, are described. Both methods are based on the formation of a ternary complex between palladium(II), eosin and the fluoroquinolone in the presence of methyl cellulose, as surfactant. Spectrophotometrically, under the optimum conditions, the ternary complexes showed an absorption maximum at 545 nm, with apparent molar absorptivities of 3.4 x 10(4) and 2.7 x 10(4) 1 mol-1 cm-1 and Sandell's sensitivities of 1.01 x 10(-2) and 1.12 x 10(-2) micrograms cm-2 for ciprofloxacin and norfloxacin, respectively. The solution of the ternary complex obeyed Beer's law in the concentration range 3-10 micrograms ml-1 for both quinolones. The proposed method was applied to the determination of the two drugs in pharmaceutical tablets. A fluorescence quenching method for the determination of both quinolones by forming this ternary complex was also investigated for the purpose of enhancing the sensitivity of the determination. The results obtained by the application of both procedures and the USP XXIII methods were in good agreement and statistical comparison by means of Student's t-test and the variance ratio F-test showed no significant differences between the three methods.

Spectrophotometric determination of etilefrine hydrochloride and ritodrine hydrochloride through nitrosation and subsequent cobalt chelation.

A simple and rapid method for the determination of etilefrine hydrochloride and ritodrine hydrochloride, either in pure form or in pharmaceutical formulations, is described. The method is based on the development of red product in presence of sodium nitrite and cobalt(II) salt in acid medium. The reaction is thought to proceed via preliminary nitrosation of the phenolic nucleus followed by formation of the chelate in presence of cobalt(II) salt. The highly colored chelates showed wavelengths of maximum absorption of 570 and 560 nm for etilefrine hydrochloride and ritodrine hydrochloride, respectively. The reaction product showed apparent molar absorptivities of 938 and 2930 L mol-1 cm-1 for etilefrine hydrochloride and ritodrine hydrochloride, respectively. A linear correlation was found between absorbance (at the lambda max) and concentration in ranges of 0.08-0.20 and 0.04-0.10 mg ml-1 for etilefrine hydrochloride and ritodrine hydrochloride, respectively. At the same time, the resulting colors were well developed within 25 min at boiling water bath temperature and were stable for more than 1 hr. Results of analysis of pure drugs and their dosage forms by the proposed method were in good agreement with either a reported derivative spectrophotometric procedure or the USP XXII method for etilefrine hydrochloride and ritodrine hydrochloride, respectively. The validity of the method was further confirmed using the standard addition method. The proposed method demonstrate high percentage of recovery with good accuracy and precision.