Noninvasive induction of angiogenesis in tissues by external suction: sequential optimization for use in reconstructive surgery.

In reconstructive surgery, tissues are routinely transferred to repair a defect caused by trauma, cancer, chronic diseases, or congenital malformations; surgical transfer intrinsically impairs metabolic supply to tissues placing a risk of ischemia-related complications such as necrosis, impaired healing, or infection. Pre-surgical induction of angiogenesis in tissues (preconditioning) can limit postsurgical ischemic complications and improve outcomes, but very few preconditioning strategies have successfully been translated to clinical practice due to the invasiveness of most proposed approaches, their suboptimal effects, and their challenging regulatory approval. We optimized a method that adopts noninvasive external suction to precondition tissues through the induction of hypoxia-mediated angiogenesis. Using a sequential approach in a rodent model, we determined the parameters of application (frequency, suction levels, duration, and interfaces) that fine-tune the balance of enhanced angiogenesis, attenuation of hypoxic tissue damage, and length of treatment. The optimized repeated short-intermittent applications of intermediate suction induced a 1.7-fold increase in tissue vascular density after only 5 days of treatment (p < 0.05); foam interfaces showed the same effectiveness and caused less complications. In a second separate experiment, our model showed that the optimized technique significantly improves survival of transferred tissues. Here we demonstrate that noninvasive external suction can successfully, safely, and promptly enhance vascularity of soft tissues: these translational principles can help design effective preconditioning strategies, transform best clinical practice in surgery, and improve patient outcomes.

Surgical procedure of extracting teeth for obtaining dental pulp for regenerative medicine in swine.

Dental pulp is a potential source of cells that can be used in cell replacement therapy for various nerve disorders, including stroke, spinal cord injury, and peripheral nerve defect. However, the validation of an animal model closely related to humans is needed in translational research. The miniature pig is a suitable experimental model in maxillofacial surgery, because its anatomical structure and size are similar to those of humans. However, the swine tooth is extremely long. The routine closed extraction procedure for harvesting dental pulp tissue causes root fracture. This report describes the details of a surgical procedure for tooth extraction. Four healthy 7-8-month-old male NIBS miniature pigs were used. Two mandibular deciduous right incisors (Di1 and Di2) were extracted in order to obtain dental pulp tissue. Gingival envelope incision with vertical-releasing incision was performed, and a full-thickness mucoperiosteal flap was made. The buccal alveolar bone was exposed and removed by osteotomy. Di1 and Di2 were extracted. Dental pulp tissue was obtained from these extracted teeth by splitting hard tissue. In this procedure, 9.8 ± 2.5 × 10(5) cells were obtained from the mandibular Di1 and Di2 (n = 4).

Familial Parkinson's disease. Alpha-synuclein and parkin.

We have reviewed recent progress in establishing the function of alpha-synuclein and parkin in relation to nigral degeneration in autosomal dominant and autosomal recessive PD. Mutations of alpha-synuclein (Ala53Thr and Ala30Pro) cause a form of autosomal dominant PD with early onset. Parkin is a novel protein expressed in the cytoplasm, including the terminal regions and Golgi apparatus. Mutations of parkin cause a form of autosomal recessive young-onset PD (ARJP). Both proteins appear to be associated with fast axonal transport. In addition, in sporadic PD, normal alpha-synuclein shows an increased tendency to self-aggregate. Thus, altered axonal transport of presynaptic proteins appears to play a crucial role in neurodegeneration in PD.

Parkin gene causing benign autosomal recessive juvenile parkinsonism.

Autosomal recessive juvenile parkinsonism (AR-JP) is an early-onset parkinsonism caused by exonic deletions or point mutations in the parkingene. The relationship between the type of the genetic defect and the clinical presentation, the response to therapy, and the evolution have not been yet determined. The authors describe a single-basepair deletion at nucleotide 202 in exon 2 of the parkin gene in a kindred with a benign clinical course.

Autosomal Recessive Juvenile Parkinsonism (AR-JP): Genetic Diagnosis.

Autosomal recessive juvenile parkinsonism (AR-JP) is a familial levodoparesponsive parkinsonism resulting from Lewy body negative degeneration of nigral neurons in the zona compacta of the substantia nigra (1-4). The first proposal for a distinct clinical entity with recessively inherited parkinsonism was made in Japan and was termed "paralysis agitans with marked diurnal fluctuations of symptoms" (1). This syndrome was later designated as autosomal recessive form of juvenile parkinsonism (2). It was subsequently found to be linked to the 17-cM region on chromosome 6q25.2-27, and the locus was recently designated Park2 (3,5). Through the study of a patient who had homozygous microdeletion of the marker D6S305 (5), the responsible gene was identified by positional cloning and was designated parkin (6). Linkage and mutation analysis to date have shown that founders of mutations in this gene are multiple and widely distributed in the world (7-13). Abnormalities in this gene, which are specific for AR-JP, include homozygous exonic deletions, small deletions, and point mutations. The presence of homozygous exonic deletions strengthens the notion that nigral neurodegeneration in AR-JP is caused by loss of function of the parkin protein.

PARKIN as a pathogenic gene for autosomal recessive juvenile parkinsonism.

Parkinson's disease is a common neurodegenerative disease with complex clinical features. Recently, we idenfied a novel gene named Parkin to be responsible for the pathogenesis of autosomal recessive juvenile parkinsonism (AR-JP). Various mutations were found in AR-JP patients of Japanese and other ethnic origins, providing a definitive evidence for the Parkin to be a causative gene for AR-JP. The predicted structure of Parkin protein and its mutation provide important clues for studying the functional role of the Parkin protein in leading to selective degeneration of nigral neurons in the brains of AR-JP patients.

Autosomal recessive early-onset parkinsonism with diurnal fluctuation: clinicopathologic characteristics and molecular genetic identification.

Autosomal recessive early-onset parkinsonism with diurnal fluctuation (AR-EPDF, syn. autosomal recessive juvenile parkinsonism, PARK2) is one of the hereditary parkinsonian syndromes. We examined subjects consisting of 43 patients from 22 families with AR-EPDF. The clinical features were relatively homogeneous, including the average age at onset of 26.1 years, beginning with dystonic gait disturbance, diurnal fluctuation of the symptoms (sleep benefit) unrelated to medication, dystonia (mainly foot dystonia), hyperactive tendon reflex, remarkable effect of levodopa and other antiparkinsonism drugs, susceptibility to dopa-induced dyskinesia, mild autonomic symptoms, absence of dementia, and slow progression of disease. Some patients had hysteric character or psychic symptoms provoked by medication. Pathologic study revealed neuronal loss in the substantia nigra pars compacta and locus coeruleus without Lewy body formation. We performed extensive molecular genetic analysis of the parkin gene in 16 families to identify a total of six different deletional mutations. In AR-EPDF loss of newly discovered 'Parkin' protein is responsible for selective degeneration of the pigmented neurons in the substantia nigra and locus coeruleus. Compared with autosomal dominant Parkinson's disease, AR-EPDF appears to be more prevalent and present in several ethnic groups.

Progress in the clinical and molecular genetics of familial parkinsonism.

Parkinson's disease (PD) is a neurodegenerative disease with clinical features resulting from deficiency of dopamine in the nigrostriatal system. Most PD cases are sporadic and the primary cause of the disease is still unknown. Recently, familial PD and parkinsonism have received much attention because these forms of the disease might provide clues to the genetic risk factors involved in the pathogenesis of idiopathic PD. To date, two causative genes, alpha-synuclein and the parkin gene, have been identified. alpha-Synuclein is involved in the pathogenesis of an autosomal dominant form of PD and constitutes a major component of the Lewy body, which is a pathological hallmark of idiopathic PD. In addition, mutations in the parkin gene have been identified as the cause of autosomal recessive juvenile parkinsonism (AR-JP). AR-JP manifests itself as a highly selective degeneration of the substantia nigra and the locus coeruleus, but without Lewy body formation. In addition to these two genes, four chromosomal loci have been linked to other forms of familial PD. Furthermore, there are a number of other pedigrees of familial PD in which linkage to known genetic loci has been excluded. Molecular cloning of these disease genes and elucidation of the function of their gene products will greatly contribute to our understanding of the pathogenesis of idiopathic PD.

Exonic deletion mutations of the Parkin gene among sporadic patients with Parkinson's disease.

Exonic deletions of the Parkin gene are common in the autosomal recessive form of juvenile parkinsonism. Here we report Parkin gene mutations among apparently sporadic Parkinson's disease (PD) patients. We screened 200 patients with PD (103 women and 97 men). The age of onset was 54.2+/-10.3years (mean+/-S.D.).Four out of the 200 patients had homozygous exonic deletions in the Parkin gene. The clinical features of these four patients were essentially the same as those of idiopathic PD. The age of onset was consistently younger (33, 38, 47 and 48years, respectively). On medication, all of them were at Hoehn and Yahr stage II or III even after 12-16years from the onset of the disease.Thus 2% of apparently sporadic PD patients in Japan have homozygous Parkin gene mutations. This positive rate was 6.3% among the patients with the age of onset below 50. Our study suggests that the prevalence of the carrier state of Parkin gene may be more than that we expected. Our study warrants further studies on Parkin gene mutations in apparently sporadic PD patients.

Autosomal-recessive juvenile parkinsonism in a Jewish Yemenite kindred: mutation of Parkin gene.

We report a Jewish family of Yemenite origin in which three brothers born from a consanguineous marriage had juvenile parkinsonism. The DNA samples from three affected brothers and one healthy brother were analyzed for the linkage to markers covering the autosomal-recessive juvenile parkinsonism (AR-JP) locus. A perfect homozygous cosegregation to the markers was found, giving a maximal lod score of 3.11 at D6S1579, D6S305, and D6S411, all of which are 0 cm apart from each other (nonparametric linkage score, 8.041; p = 0.000977). Exon 3 of the Parkin gene was homozygously deleted in all patients. The AR-JP gene also exists in the Jewish population.

[A Parkin gene (PARK 2) and Parkinson's disease].

We identified a PARK 2 (AR-JP) family with a patient presenting with homozygous deletion of D 6 S305--a marker within the 17cM region for PARK 2 locus. Markers surrounding D 6 S305 which are mapped 0 cM apart from D 6 S305, were not deleted, indicating that PARK 2 gene is located extremely close to D 6 S305. Exon search in the inserts with average size of 100 kb of BAC clones, which harbor D 6 S305, led us to find the exonic sequences which was subsequently proved to be exon 7 of the Parkin gene. From this exon sequences, full-length cDNA was isolated, and BAC contig covering Parkin gene was generated. Homozygous deletions or frame-shift mutations in the Parkin gene were found in the patients with AR-JP/PARK 2, revealing that a loss-of-function of Parkin gene is responsible for AR-JP/PARK 2. Our findings indicate that constant production of Parkin protein is essentially required for maintaining the survival of nigral neurons. One attractive hypothesis is that Parkinson's disease and AR-JP/PARK 2 might share a common effector pathway for nigral neuronal death. In this scenario, as PARK 2 is not accompanied with Lewy body formation. Parkin might act at or downstream of synuclein aggregation, which has been recently implicated as a trigger event for neuronal death in Parkinson's disease. In any case, identification of functional targets of Parkin protein will give us an important clue to identify downstream events of neuronal death which is activated by inclusion body formation.

Polymorphism in the parkin gene in sporadic Parkinson's disease.

We report polymorphism of the parkin gene in 160 sporadic Parkinson's disease (PD) patients and controls. Three polymorphisms were found: a G-to-A transition in exon 4 (S/N167), a C-to-T transition in exon 10 (R/W366), and a G-to-C transition in exon 10 (V/L380). Genotype distributions and allele frequencies of S/N167 and V/L380 did not differ significantly between the two groups. The R/W366 allele frequency was significantly lower in PD patients (1.2 vs 4.4%). The level of protection from PD provided by this allele was 3.60 (95% CI; range, 0.45-6.50), suggesting that it may be a protective factor against PD.

Immunohistochemical and subcellular localization of Parkin protein: absence of protein in autosomal recessive juvenile parkinsonism patients.

Autosomal recessive juvenile parkinsonism (AR-JP) is a distinct clinical entity characterized by a selective degeneration of nigral neurons. Recently, the parkin gene responsible for AR-JP has been identified. Now, we report the subcellular localization of Parkin protein in patients with AR-JP or Parkinson's disease (PD) and in controls by immunoblotting and immunohistochemistry using antibodies raised against the Parkin molecule. Parkin protein was absent in all regions of the brains of patients with AR-JP. Parkin protein was not decreased in the brains of sporadic PD patients. Immunoreactivity was detected in a few Lewy bodies. Parkin protein was located in both the Golgi complex and cytosol.

Positional cloning of the autosomal recessive juvenile parkinsonism (AR-JP) gene and its diversity in deletion mutations.

Autosomal recessive juvenile parkinsonism (AR-JP) is a distinct clinical and genetic entity characterized by highly selective neuronal cell death in the substantia nigra and the locus coeruleus with no Lewy body formation. We succeeded in positional cloning of the AR-JP gene by screening the Keio BAC library with a microsatellite marker, D6S305, which is located AR-JP locus (6q25.2-q27). The gene was named as parkin; parkin consists of 12 exons spanning about 1Mb with 1395bp coding sequence. Patients with AR-JP showed various deletions in 14 Japanese families and two different types of point mutations in two Turkish families. AR-JP appears to have world-wide distribution.

Molecular genetic analysis of a novel Parkin gene in Japanese families with autosomal recessive juvenile parkinsonism: evidence for variable homozygous deletions in the Parkin gene in affected individuals.

Autosomal recessive juvenile parkinsonism (AR-JP) is a distinct clinical and genetic entity characterized by selective degeneration of nigral dopaminergic neurons and young-onset parkinsonism with remarkable response to levodopa. Recently, we mapped the gene locus for AR-JP to chromosome 6q25.2-q27 by linkage analysis and we identified a novel large gene, Parkin, consisting of 12 exons from this region; mutations of this gene were found to be the cause of AR-JP in two families. Now we report results of extensive molecular analysis on 34 affected individuals from 18 unrelated families with AR-JP. We found four different homozygous intragenic deletional mutations, involving exons 3 to 4, exon 3, exon 4, and exon 5 in 10 families (17 affected individuals). In addition to the exonic deletions, we identified a novel one-base deletion involving exon 5 in two families (2 affected individuals). All mutations so far found were deletional types in which large exonic deletion accounted for 50% (17 of 34) and the one-base deletion accounted for 6% (2/34); in the remaining, no homozygous mutations were found in the coding regions. Our findings indicate that loss of function of the Parkin protein results in the clinical phenotype of AR-JP and that subregions between introns 2 and 5 of the Parkin gene are mutational hot spots.

A loss-of-function mechanism of nigral neuron death without Lewy body formation: autosomal recessive juvenile parkinsonism (AR-JP).

We present a genetic model of selective nigral neuron death without Lewy body formation--autosomal recessive juvenile parkinsonism (AR-JP). Mapping of AR-JP gene to a single recessive locus on chromosome 6q25.2-27 is a strong indication of the idea that a loss-of-function mutation of a single gene is sufficient to cause selective nigral neuron death. The absence of Lewy body formation in AR-JP is important, because most cases of neuron death caused by loss-of-function mechanism are not accompanied by inclusion body. Our findings clearly indicate that nigral cells are critically dependent for their survival on the function of AR-JP protein. This raises an important question of whether or not AR-JP protein contributes to the downstream pathway of cell death in Parkinson's disease.

[A 64-year-old woman with progressive gait disturbance and dementia for one year].

We report a 64-year-old Japanese woman who died one year after the onset of progressive gait disturbance and dementia. She noted a difficulty in holding a glass and hand tremor in June of 1996 when she was 63 years old. In July of 1996, she tended to lean toward left when she walked. She also noted truncal titubation. In November of 1996, she started to have visual hallucination and delusion in which she said "I see something is flying on the wall.", "Somebody has come into my room", and things like that. She was admitted to our service on November 22, 1996. On admission, she was alert and general physical examination was unremarkable. Neurologic examination revealed disturbance in recent memory. Hasegawa's dementia rating scale was 22/30. She showed vivid visual hallucination with colors in which she saw faces of dwarfs and angels, a space ship, and others. Higher cerebral functions were normal. She showed left oculomotor palsy which was a sequel of an aneurysm and subarachnoid hemorrhage nine years before. Otherwise cranial nerves were unremarkable. She showed ataxic gait, limb ataxia, truncal titubation, and postural hand tremor. She had no weakness and no muscle atrophy. Deep tendon reflexes were within normal limits. Plantar response was flexor. Sensation was intact. Laboratory examination was also unremarkable. Complete survey for occult malignancy was negative. CSF was under a normal pressure and cell count was 1/microliter, total protein 27 mg/dl, and sugar 68 mg/dl. Cranial CT scan was unremarkable. MRI was not obtained because of the presence of an aneurysm clip in the left internal carotid-posterior communication artery junction. She showed progressive deterioration in her mental function. By January 1997, she became unable to stand or walk with marked dementia. Repeated CSF exams and cranial CT scans were unremarkable. She suffered from several episodes of aspiration pneumonia. A trial of three days methylprednisolone pulse therapy was given starting on March 7, 1997, which was of no effect on her neurologic status. On March 28, 1997, she was intubated because of acute respiratory distress syndrome. In April 2, her body temperature rose to 38 degrees C. On April 9, 1997, her blood pressure dropped and resuscitation was unsuccessful. She was pronounced dead on the same day. The patient was discussed in a neurologic CPC and the chief discussant arrived at the conclusion that the patient had primary leptomeningeal lymphoma. Other possibilities entertained among the audience included brain stem encephalitis of unknown type, carcinomatous cerebellar degeneration plus limbic encephalitis, Creutzfeldt-Jakob disease, thalamic degeneration, and progressive multifocal leukoencephalopathy. Post-mortem examination revealed thickening and clouding of the leptomeninges; Gram-positive diplococci were found in the leptomeninges. This meningitis appeared to have been an complication in the terminal stage of her illness. Microscopic examination revealed astrocytosis in the midbrain tegmentum. Cerebral cortices showed only mild astrtocytosis. No cerebellar atrophy was seen and Purkinje cells were retained which excluded paraneoplastic cerebellar degeneration. Neuropathologic diagnosis was bacterial meningitis, however, the presence of brain stem encephalitis prior to the onset of bacterial meningitis could not be excluded. It is interesting to note that the diagnosis of the primary neurologic disease of this patient was not easy even after autopsy. As autopsy permission was obtained only for the brain, it was not clear whether or not this patient had an occult malignancy somewhere in her body, however, there was no evidence to indicate paraneoplastic degeneration of the central nervous system. As the patient did not have meningeal signs until one month before her death, it is difficult to ascribe her entire neurologic problems to her meningitis. Finally, her visual hallucination was vivid and colorful; we thought this might have been

Mitochondrial dysfunction in Parkinson's disease.

This review discusses the etiology and pathogenesis of Parkinson's disease (PD). Mitochondrial respiratory failure and oxidative stress appear to be two major contributors to nigral neuronal death in PD. Complex I deficiency has been reported by several groups and appears to be one of the basic abnormalities responsible for mitochondrial failure. The principal question is whether or not complex I deficiency is primary or secondary. The second question is whether or not complex I deficiency is localized in the nigrostriatal system or is systemically present. It is our impression that complex I deficiency is not the primary cause but that its deficiency appears to be systemic. The primary cause may be the combination of genetic background and potential nigral neurotoxins. Exposure of nigral neurons to a high risk for oxidative damage because of its high dopamine content may be the reason for more pronounced nigral complex I deficiency compared to systemic organs. Oxidative stress and mitochondrial failure produce a vicious cycle in nigral neurons. To explore the genetic risk factors of sporadic PD, studies on familial PD and parkinsonism are important. Recently, an autosomal dominant form of familial PD was found to be caused by point mutations of the alpha-synuclein gene, and an autosomal recessive familial parkinsonism was mapped to the long arm of chromosome 6 near the Mn-SOD gene locus. Information obtained in these familial cases will contribute to the research on sporadic PD.