Jorge Rodrigues

Long-term functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp.

We have recently described the sequence of functional and morphologic changes occurring after a standardized sciatic nerve crush injury. An 8-week post-injury time was used because this end point is the far most used. Unexpectedly, both functional and morphological data revealed that animals had still not recovered to normal pre-injury levels. Therefore, the present study was designed in order to prolong the observation up to 12 weeks. Functional recovery was evaluated using sciatic functional index (SFI), static sciatic index (SSI), extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. In addition, quantitative morphology was carried out on regenerated nerve fibers. A full functional recovery was predicted by SFI/SSI, EPT and WRL but not all ankle kinematics parameters. Moreover, only two morphological parameters (myelin thickness/axon diameter ratio and fiber/axon diameter ratio) returned to normal values. Data presented in this paper provide a baseline for selecting the adequate end-point and methods of recovery assessment for a rat sciatic nerve crush study and suggest that the combined use of functional and morphological analysis should be recommended in this experimental model.

Effects of collagen membranes enriched with in vitro-differentiated N1E-115 cells on rat sciatic nerve regeneration after end-to-end repair

Peripheral nerves possess the capacity of self-regeneration after traumatic injury but the extent of regeneration is often poor and may benefit from exogenous factors that enhance growth. The use of cellular systems is a rational approach for delivering neurotrophic factors at the nerve lesion site, and in the present study we investigated the effects of enwrapping the site of end-to-end rat sciatic nerve repair with an equine type III collagen membrane enriched or not with N1E-115 pre-differentiated neural cells. After neurotmesis, the sciatic nerve was repaired by end-to-end suture (End-to-End group), end-to-end suture enwrapped with an equine collagen type III membrane (End-to-EndMemb group); and end-to-end suture enwrapped with an equine collagen type III membrane previously covered with neural cells pre-differentiated in vitro from N1E-115 cells (End-to-EndMembCell group). Along the postoperative, motor and sensory functional recovery was evaluated using extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. After 20 weeks animals were sacrificed and the repaired sciatic nerves were processed for histological and stereological analysis. Results showed that enwrapment of the rapair site with a collagen membrane, with or without neural cell enrichment, did not lead to any significant improvement in most of functional and stereological predictors of nerve regeneration that we have assessed, with the exception of EPT which recovered significantly better after neural cell enriched membrane employment. It can thus be concluded that this particular type of nerve tissue engineering approach has very limited effects on nerve regeneration after sciatic end-to-end nerve reconstruction in the rat.

Use of PLGA 90:10 Scaffolds Enriched with In Vitro–Differentiated Neural Cells for Repairing Rat Sciatic Nerve Defects

Poly(lactic-co-glycolic acid) (PLGA) nerve tube guides, made of a novel proportion (90:10) of the two polymers, poly(L-lactide): poly(glycolide) and covered with a neural cell line differentiated in vitro, were tested in vivo for promoting nerve regeneration across a 10-mm gap of the rat sciatic nerve. Before in vivo testing, the PLGA 90:10 tubes were tested in vitro for water uptake and mass loss and compared with collagen sheets. The water uptake of the PLGA tubes was lower, and the mass loss was more rapid and higher than those of the collagen sheets when immersed in phosphate-buffered saline (PBS) solution. The pH values of immersing PBS did not change after soaking the collagen sheets and showed to be around 7.4. On the other hand, the pH values of PBS after soaking PLGA tubes decreased gradually during 10 days reaching values around 3.5. For the in vivo testing, 22 Sasco Sprague adult rats were divided into four groups--group 1: gap not reconstructed; group 2: gap reconstructed using an autologous nerve graft; group 3: gap reconstructed with PLGA 90:10 tube guides; group 4: gap reconstructed with PLGA 90:10 tube guides covered with neural cells differentiated in vitro. Motor and sensory functional recovery was evaluated throughout a healing period of 20 weeks using sciatic functional index, static sciatic index, extensor postural thrust, withdrawal reflex latency, and ankle kinematics. Stereological analysis was carried out on regenerated nerve fibers. Both motor and sensory functions improved significantly in the three experimental nerve repair groups, although the rate and extent of recovery was significantly higher in the group where the gap was reconstructed using the autologous graft. The presence of neural cells covering the inside of the PLGA tube guides did not make any difference in the functional recovery. By contrast, morphometric analysis showed that the introduction of N1E-115 cells inside PLGA 90:10 tube guides led to a significant lower number and size of regenerated nerve fibers, suggesting thus that this approach is not adequate for promoting peripheral nerve repair. Further studies are warranted to assess the role of other cellular systems as a foreseeable therapeutic strategy in peripheral nerve regeneration.

Protective effects of a catechin-rich extract on the hippocampal formation and spatial memory in aging rats

Green tea (GT) displays strong anti-oxidant and anti-inflammatory properties mostly attributed to (-)-epigallocatechin-3-gallate (EGCG), while experiments focusing on other catechins are scarce. With the present work we intended to analyze the neuroprotective effects of prolonged consumption of a GT extract (GTE) rich in catechins but poor in EGCG and other GT bioactive components that could also afford benefit. The endpoints evaluated were aging-induced biochemical and morphological changes in the rat hippocampal formation (HF) and behavioral alterations. Male Wistar rats aged 12 months were treated with GTE until 19 months of age. This group of animals was compared with control groups aged 19 (C-19M) or 12 months (C-12M). We found that aging increased oxidative markers but GTE consumption protected proteins and lipids against oxidation. The age-associated increase in lipofuscin content and lysosomal volume was also prevented by treatment with GTE. The dendritic arborizations of dentate granule cells of GTE-treated animals presented plastic changes accompanied by an improved spatial learning evaluated with the Morris water maze. Altogether our results demonstrate that the consumption of an extract rich in catechins other than EGCG protected the HF from aging-related declines contributing to improve the redox status and preventing the structural damage observed in old animals, with repercussions on behavioral performance.

Neural cell transplantation effects on sciatic nerve regeneration after a standardized crush injury in the rat.

The goal of the present study was to assess whether in vitro‐differentiated N1E‐115 cells supported by a collagen membrane would enhance rat sciatic nerve regeneration after a crush injury. To set up an appropriate experimental model for investigating the effects of neural cell transplantation, we have recently described the sequence of functional and morphologic changes occurring after a standardized sciatic nerve crush injury with a nonserrated clamp. Functional recovery was evaluated using the sciatic functional index, the static sciatic index, the extensor postural thrust, the withdrawal reflex latency, and ankle kinematics. In addition, histomorphometric analysis was carried out on regenerated nerve fibers by means of the 2D‐disector method. Based on the results of the EPT and of some of the ankle locomotor kinematic parameters analyzed, the hypothesis that N1E‐115 cells may enhance nerve regeneration is partially supported although histomorphometry disclosed no significant difference in nerve fiber regeneration between the different experimental groups. Therefore, results suggest that enrichment of equine type III collagen membrane with the N1E‐115 cellular system in the rat sciatic nerve crush model may support recovery, at least in terms of motor function. The discrepancy between functional and morphological results also suggests that the combined use of functional and morphological analysis should be recommended for an overall assessment of recovery in nerve regeneration studies.

Assessment of the Potential of Bonelike® Graft for Bone Regeneration by Using an Animal Model

BonelikeÒ graft that mimics the inorganic composition of bone tissue has been developed and characterized over the last decade. To evaluate the osteoconductivity of BonelikeÒ two granule size ranges, one ranging from 150-250µm and the other from 250-500µm were implanted in the femurs of New Zealand White rabbits, aiming at being clinically used in different medical applications, such as dentistry and orthopaedics. In order to facilitate the medical application of the BonelikeÒ graft the use of a commercially available polymeric vehicle was also analyzed. Radiological examination, histological studies and scanning electron microscopy (SEM) analyses revealed that the surface of Bonelike® granules was almost completely surrounded by new bone formation after 12 weeks of implantation, which proves its highly osteoconductive behaviour.

Inner ear involvement in fabry disease: Clinical and audiometric evaluation of a large cohort of patients followed in a reference centre

BACKGROUND Fabry disease (FD) is a lysosomal storage disorder (LSD) that involves the cochleovestibular system. Tinnitus and progressive sensorineural hearing loss are frequent complains. A stabilization of hearing function has been reported with enzyme replacement therapy (ERT). This study aims to characterize the inner ear involvement, identify factors associated to hearing loss and evaluate the effect of ERT on the hearing function of FD patients. METHODS We reviewed the clinical records of patients with confirmed diagnosis of FD followed in a Reference Centre on LSD in the North of Portugal. RESULTS We included a total of 122 patients with a mean age of 47.1 ± 17.6 years and 48.3% males. Hearing loss was reported by 26.2% of the patients and 23.0% mentioned tinnitus. Pure tone audiometry revealed sensorineural hearing loss in 36.9% of the cases. FD patients presented worse age-adjusted hearing thresholds in all analysed frequencies compared to the normal population (p = .001). Patients with hearing loss presented a significantly higher value of microalbuminuria (p = .001) and a higher frequency of acroparesthesias (p = .032). Patients presented a comparable hearing level one year after starting ERT (p = .384). CONCLUSIONS In FD, hearing loss is common and age-matched hearing thresholds by frequency are worse than in the general population. Hearing loss was associated to the presence of acroparesthesias and higher values of microalbuminuria. Hearing loss stabilized in patients under ERT. A careful cochleo-vestibular evaluation should be part of the clinical assessment of FD.

Surgically-Treated Locoregionally Advanced Hypopharyngeal Cancer: Outcomes

Introduction  Hypopharyngeal tumors are head and neck malignancies associated with a great mortality rate, and the treatment of advanced lesions constitutes a challenging problem. Pharyngolaryngectomy continues to be the gold standard treatment modality for locally-advanced diseases, and it is currently used as the primary treatment or in cases of relapse after an organ preservation strategy. Objective  This study aims to compare the survival rates of patients with advanced hypopharyngeal tumors treated with pharyngolaryngectomy as a primary or salvage option, and identify possible prognostic factors. Methods  All patients with advanced hypopharyngeal squamous cell carcinomas who performed pharyngolaryngectomy between 2007 and 2014 were reviewed retrospectively. Results  A total of 87 patients fulfilled the aforementioned criteria, and the sample had a mean age of 57.2 years and a male predominance of 43:1. The tumors were located in the pyriform sinus walls (81 tumors), in the posterior pharyngeal wall (4 tumors) and in the postcricoid region (2 tumors). A total of 60 patients underwent surgery as the primary treatment option, and 27 were submitted to salvage pharyngolaryngectomy after a previous treatment with chemoradiotherapy or radiotherapy. The 5-year overall survival was of 25.9%, the 5-year disease-free survival was of 24.2%, and the disease-specific survival was of 29.5%. Conclusion  The patients treated with pharyngolaryngectomy as the primary option revealed a better 5-year-disease free survival than the patients who underwent the salvage surgery (35.8% versus 11.7% respectively; p  < 0.05). The histopathological criteria of capsular rupture of the lymph nodes (30.1% versus 19.8% respectively for the primary and salvage groups; p  < 0.05) and vascular invasion (30.5% versus 22.5% respectively; p  < 0.05) reduced the 5-year disease-free survival. Pharyngolaryngectomy as the primary intent revealed a lower local recurrence rate than the salvage surgery (40.6% versus 83.3% respectively; p  < 0.05).

Ossiculoplasty in chronic otitis media: Surgical results and prognostic factors of surgical success

BACKGROUND AND OBJECTIVES The goal of ossiculoplasty is to improve hearing. Successful ossiculoplasty depends on several factors. This retrospective study was carried out to analyze hearing results of ossiculoplasty in ears with chronic otitis media (COM) and evaluate clinical outcomes and factors predictive of hearing improvement. SUBJECTS AND METHODS We reviewed the results of 153 patients with COM (with cholesteatoma (COMC) and without cholesteatoma (COMWC)) who underwent ossiculoplasty between January of 2002 to December of 2011. Several potential prognostic factors were evaluated: cholesteatoma present vs absent; type of surgical procedure, state of the middle ear mucosa, state of the ossicular chain, type of prosthesis. RESULTS We analyzed 153 ossiculoplasties: 96 patients presented COMWC and 57 patients presented COMC. The ossiculoplasties were performed using autologous ossicles for the most part. All ossiculoplasties were carried out in one-stage surgery. In 38% of cases ossiculoplasty was combined with mastoidectomy; in the remaining 62% of cases, ossiculoplasty was performed without mastoidectomy. Ossiculoplasty was successfully achieved in 113 patients (74%). The presence of the stapes superstructure and normal mucosa were significant predictive factors of surgical success. CONCLUSION The majority of the ossiculoplasties improved hearing status satisfactorily. Multivariate analysis should be performed to investigate prognostic factors of favorable short-term hearing outcomes after ossiculoplasty. Better knowledge of these predictive factors may contribute to the surgeons judgment and the information given to patients.

Chapter 112 – Green Tea and Protection of the Brain Against Aging: Structural Aspects

Aging leads to oxidative imbalances in the central nervous system, predisposing it to neurodegenerative diseases. The brain is vulnerable to oxidative events involved in the formation of lipofuscin, a non-degradable pigment formed in lysosomes and considered a biomarker of neuronal lesion. The polyphenols present in green tea have beneficial effects in several models of neurotoxicity, and the prolonged consumption of this beverage was able to decrease lysosomal volume, reduce the deposition of lipofuscin in neuronal cytoplasm and diminish mitochondrial oxidative events in rodents during aging. Furthermore, it maintained the cognitive performance dependent on the structural integrity of the hippocampal formation in aged animals. The modulation of cellular signaling systems related to anti-oxidant and anti-inflammatory defenses and neuronal survival is probably related to the marked neuroprotective action of green tea polyphenols. These properties suggest that green tea can be helpful in delaying the effects of aging in the brain and preventing neurodegenerative diseases.