Hachiro Nakagawa

Degeneration of retinal neuronal processes and pigment epithelium in the early stage of the streptozotocin-diabetic rats.

Early pathological and electro‐physiological changes of the retina in the streptozotocin (STZ)‐diabetic rats were investigated through optical and electron microscopy in two strains and electro‐retinography in one strain. In Sprague–Dawley (SD) rats 1 month after the onset of diabetes, the thickness of the inner plexiform layer (IPL) and photoreceptor segment layer (PSL) was significantly reduced by 9.9% and 18.9%, respectively (P < 0.01, P < 0.05). In Brown–Norway (BN) rats STZ‐diabetic for 1 month, the thickness of the IPL was also significantly reduced by 15.7% (P < 0.05). Cytochemical study using peanut agglutinin (PNA), a lectin binding selectively to the cone photoreceptor‐associated domains of the inter‐photoreceptor matrix, revealed a marked reduction in intensity, number and length of the PNA‐binding cone photoreceptors. Electron microscopy showed deepened hollows in the basal infoldings of the retinal pigment epithelium (RPE) of STZ‐rats diabetic for 1 month and large concavities into the cytoplasm in STZ‐rats diabetic for 6 months. Blood vessels in the retina and choroid were unremarkable. Single‐flash electro‐retinogram revealed a reduction in the amplitudes of a‐ and b‐waves of electro‐retinogram (ERG) of 1 month STZ BN rats (P < 0.05). These findings indicate that the degeneration of rods/cones in the PSL and RPE are the most prominent pathological alteration sites in the early stage of diabetic rats.

The N-terminal active centre of human angiotensin-converting enzyme degrades Alzheimer amyloid β-peptide

We reported recently that angiotensin‐converting enzyme (ACE) significantly degraded amyloid β‐peptide (Aβ) to inhibit aggregation and cytotoxicity of Aβ in PC12h cells in vitro. On the other hand, others reported that ACE had two domains with highly homologous active centres, the N‐domain and C‐domain, but that they differed in their characteristics such as optimum chloride ion concentration, inhibition kinetics for various ACE inhibitors and rate of hydrolysis for many substrates. The aim of this study was to determine the specific ACE domain primarily responsible for degradation of Aβ. For this purpose, a series of ACE recombinant proteins, each containing only one intact domain, was constructed and expressed in COS7. Our results showed that all ACE recombinant proteins obtained were enzymatically active in terms of angiotensin I cleavage. However, inhibition of Aβ aggregation and cytotoxicity of the N‐domain were higher than those of the C‐domain. Reverse‐phase high‐performance liquid chromatography analyses confirmed that the N domain degraded Aβ. Our results indicate that the N domain of ACE is primarily responsible for the degradation of Aβ.

Increased interleukin-6 of skin and serum in amyotrophic lateral sclerosis

Studies of skin in amyotrophic lateral sclerosis (ALS) have demonstrated morphological and biochemical alterations. Interleukin-6 (IL-6) has been suggested to have a trophic effect in nerve cells and to have a direct pathogenic role in neurodegenerative central nervous system (CNS) disorders. However, little is known concerning IL-6 in ALS patients. We examined IL-6 immunoreactivity of biopsy specimens of skin and measured serum IL-6 levels from 11 ALS patients and 11 diseased control subjects. IL-6 immunoreactivity was markedly positive in the epidermis and dermal blood vessels and glands and was moderately positive in the reticular dermis in all ALS patients. These optical densities for IL-6 immunoreactivity in ALS patients were significantly higher than in control subjects, and were significantly increased with duration of illness. Serum IL-6 levels were detected in 8 (73%) of 11 ALS patients compared with only 1 (9%) of 11 diseased control subjects. Serum IL-6 levels were significantly correlated with duration of illness in ALS patients and immunoreactivity of IL-6 of the epidermis. These data suggest that the increased levels of serum IL-6 may reflect an increased IL-6 immunoreactivity of skin in ALS patients.

Increased expression of insulin-like growth factor I in skin in amyotrophic lateral sclerosis

OBJECTIVES Insulin-like growth factor I (IGF-I) has potent effects on motor neuron survival and is being studied as a possible therapeutic agent for ALS. However, little is known concerning IGF-I in the skin of patients with amyotrophic lateral sclerosis (ALS). The aim was to evaluate IGF-I immunoreactivity of skin in patients with ALS. METHODS IGF-I immunoreactivity of skin from 18 patients with ALS and 16 controls was examined. RESULTS IGF-I immunoreactivity was markedly positive in the epidermis and dermal blood vessels and glands and was moderately positive in the reticular dermis in all patients with ALS. On the other hand, the epidermis and dermal blood vessels and glands and the reticular dermis showed a weak IGF-I immunoreactivity in controls. The optical density for IGF-I immunoreactivity of the epidermis and dermal blood vessels and glands, and the reticular dermis in patients with ALS was significantly higher than in diseased controls, and was significantly increased with duration of illness. CONCLUSIONS These data suggest that a metabolic alteration of IGF-I may take place in the skin of patients with ALS.

Distinctive expression of midkine in the repair period of rat brain during neurogenesis: immunohistochemical and immunoelectron microscopic observations.

Distinctive expression of midkine (MK) was observed during the repair period of fetal brain neuroepithelium. MK is a heparin‐binding growth factor that occurs as a product of a retinoic acid‐inducible gene, and has a molecular mass of 13 kDa. MK expression was examined immunohistochemically and by immunoelectron microscopy during a period of repair in developing rat brain at the neurogenesis stage. Injury was induced in rat fetuses by transplacental administration of ethylnitrosourea (ENU) on embryonic Day (E) 16, and histological changes were examined up to 48 hr thereafter (i.e., up to E 18). In normal rat fetuses, MK immunostaining was observed in the cytoplasm and radial and horizontal processes of all cells in the neuroepithelium (NE), subventricular zone (SV), and intermediate zone (IMZ). In ENU‐administered brains, cells in the NE, SV, and IMZ were damaged severely, especially 16–24 hr after ENU administration. The remaining neuroepithelial cells, with the exception of those in M‐phase and the tips of processes at the ventricular surface, were negative for MK immunohistochemistry 16–24 hr after the administration of ENU. Forty‐eight hours after the administration, the cytoplasm and processes of cells in the NE, SV, and IMZ were MK immunopositive. Our previous data reported that the cell cycle of most NE cells is synchronized to the S‐phase 16 hr after ENU administration and to the M‐phase at 24 hr, and many NE cells were recovered 48 hr after ENU administration. The previous results taken together with the present results indicate that: (1) MK expression does not increase during the repair period of the NE, being different from adults; (2) MK expression is likely to be suppressed at S‐phase according to the condition of the NE; and (3) MK expression is not essential for every cell cycle phase of NE cells; but (4) is necessary to maintain the M‐phase of NE cells.

Decrease in the Ciliary Neurotrophic Factor of the Spinal Cord in Amyotrophic Lateral Sclerosis

Ciliary neurotrophic factor (CNTF), a potent survival factor in spinal motoneurons of embryonic chick and rat, is currently being investigated in humans as a treatment for amyotrophic lateral sclerosis (ALS). However, its physiological and pathological activities in ALS remain unclear. We measured CNTF contents in the cervical enlargement of the spinal cord from 9 ALS patients and 12 age-matched control subjects using a sensitive enzyme-linked immunoassay. CNTF genotypes were determined by the polymerase chain reaction-restriction fragment length polymorphism method. In control subjects, there were 8 homozygotes and 4 heterozygotes, while in ALS patients there were 6 and 3, respectively. In both homozygotes and heterozygotes, CNTF expression in the spinal cord from ALS patients tended to decrease compared to control subjects. In homozygotes, the decrease was significant (p < 0.05). Concerning the regional concentrations of CNTF, in homozygotes, CNTF contents in the lateral corticospinal tract were markedly lower (p < 0.001) in ALS patients than in controls. The decrease in CNTF expression in the lateral corticospinal tract of the spinal cord from ALS patients may be a feature of ALS and could be related to motor neuron loss.

Expression of leukaemia inhibitory factor in skin of patients with amyotrophic lateral sclerosis

One of the remarkable features of patients with amyotrophic lateral sclerosis (ALS) is absence of bedsores. Our previous reports have shown morphological and biochemical alterations in the skin of patients with ALS. , 3 L e u k a e m i a inhibitory factor (LIF) belongs to a group of cytokines including CNTF, IL-6, CT-1, OM, and IL-11. All the group members have different biological effects, but use gp130 signal-transducing subunit as common receptor. LIF is located on the long arm of chromosome 22, adjacent to the sporadic ALS-related NF-H gene. LIF affects survival and maintenance of both motor and sensory neurons, and promotes the differentiation of neuronal and astrocytic precursors within the developing spinal cord. LIF also acts as a myogenic growth factor, stimulating muscle regeneration. L I F is significantly upregulated in spinal cord when neurons are damaged by BOAA toxin, an excitatory aminoacid associated with a form of ALS. LIF is now under clinical investigation as a treatment for ALS. However, little is known about whether expression of LIF is altered in the skin of patients with ALS. Skin biopsy samples were taken from the left upper arm of ten patients with ALS (six men, mean age 56·2 years [range 50–72]) and from ten controls with other neurodegenerative diseases matched for sex and age (51·7 years [48–68]). Informed consent was obtained from all patients. Routine formalin-fixed paraffin-embedded 6 m sections were immunostained according to standard techniques. The sections were incubated with goat anti-LIF antibody (Santa Crus). After washing in phsophate-buffered saline, biotinylated anti-goat IgG (Vector) was applied. The sections were stained by ABC kit (Vector). The immunoreactive intensity was quantified with an image-analysis system (Carl Z e i s s ) . L I F immunoreactivity was strongly positive in the epidermis and in some blood vessels and glands of the without radiation, Second, when the group of gross totally resected carcinomas was further categorised into infants and older patients, the outcome after radiation remained significantly better in the older group (p=0·007), and a tendency to better survival with radiation was also seen in the smaller group of infants (p=0·659, not significant). This finding suggests that radiation improves survival in carcinomas and that an age bias does not exist. We conclude that patients with choroid plexus carcinoma should receive radiotherapy as an adjuvant treatment after surgery, if the patient is not too young.