Metabolomics study of cerebrospinal fluid from diabetic rats with cognitive impairment simultaneously treated with Panax quinquefolius and Acorus gramineus.

A metabolomics approach was used to explore the effects of Panax quinquefolius (PQ) and Acorus gramineus (AG) on learning and memory in rats with diabetic-induced cognitive impairment. Thirty Wistar rats were divided into three groups, namely, the normal group, model group, and PQ-AG group (PQ-AG group, 1.80 g/kg/d). Diabetes was induced by intraperitoneal injection of streptozotocin (65 mg/kg). Cerebrospinal fluid (CSF) was collected via cisterna magna puncture, and the Morris water maze method was used to evaluate learning and memory in rats after 11 weeks of PQ-AG treatment. Metabolic profiling of CSF samples was performed by using UPLC-Q-TOF-MS. Compared with the normal group, the escape latency of the Morris water maze was significantly prolonged in model group rats after 12 weeks (p < 0.01). Compared with the model group, however, the escape latency was significantly shortened in PQ-AG group rats (p < 0.05). In multivariate statistical analysis, we identified 33 potential biomarkers, and six biomarkers were altered by PQ-AG. These biomarkers were involved in the metabolism of pyrimidine; nicotinate, and nicotinamide; glycine, serine, and threonine; and ascorbate and aldarate. Taken collectively, our results indicate that PQ-AG can attenuate diabetic-induced cognitive impairment by affecting a variety of metabolic pathways. Our results provide an experimental basis for studying the mechanism of action of PQ-AG.

Exendin-4 Reverses Biochemical and Functional Alterations in the Blood-Brain and Blood-CSF Barriers in Diabetic Rats.

Diabetes mellitus (DM) is a metabolic disorder associated with micro- and macrovascular alterations that contribute to the cognitive impairment observed in diabetic patients. Signs of breakdown of the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) have been found in patients and animal models of DM. Breakdown of the BBB and BCSFB can lead to disruptions in cerebral homeostasis and eventually neural dysfunction and degeneration. However, our understanding of the biochemistry underlying barrier protein modifications is incomplete. Herein, we evaluated changes in the levels of specific proteins in the BBB (occludin, claudin-5, ZO-1, and aquaporin-4) and BCSFB (claudin-2 and aquaporin-1) in the hippocampus of diabetic rats, and we also investigated the functional alterations in these barriers. In addition, we evaluated the ability of exendin-4 (EX-4), a glucagon-like peptide-1 agonist that can cross the BBB to reverse the functional and biochemical modifications observed in these animals. We observed a decrease in BBB proteins (except ZO-1) in diabetic rats, whereas the EX-4 treatment recovered the occludin and aquaporin-4 levels. Similarly, we observed a decrease in BCSFB proteins in diabetic rats, whereas EX-4 reversed such changes. EX-4 also reversed alterations in the permeability of the BBB and BCSFB in diabetic rats. Additionally, altered cognitive parameters in diabetic rats were improved by EX-4. These data further our understanding of the alterations in the central nervous system caused by DM, particularly changes in the proteins and permeability of the brain barriers, as well as cognitive dysfunction. Furthermore, these data suggest a role for EX-4 in therapeutic strategies for cognitive dysfunction in DM.

Impairment of the glymphatic system after diabetes.

The glymphatic system has recently been shown to clear brain extracellular solutes and abnormalities in glymphatic clearance system may contribute to both initiation and progression of neurological diseases. Despite that diabetes is known as a risk factor for vascular diseases, little is known how diabetes affects the glymphatic system. The current study is the first investigation of the effect of diabetes on the glymphatic system and the link between alteration of glymphatic clearance and cognitive impairment in Type-2 diabetes mellitus rats. MRI analysis revealed that clearance of cerebrospinal fluid contrast agent Gd-DTPA from the interstitial space was slowed by a factor of three in the hippocampus of Type-2 diabetes mellitus rats compared to the non-DM rats and confirmed by florescence imaging analysis. Cognitive deficits detected by behavioral tests were highly and inversely correlated to the retention of Gd-DTPA contrast and fluorescent tracer in the hippocampus of Type-2 diabetes mellitus rats. Type-2 diabetes mellitus suppresses clearance of interstitial fluid in the hippocampus and hypothalamus, suggesting that an impairment of the glymphatic system contributes to Type-2 diabetes mellitus-induced cognitive deficits. Whole brain MRI provides a sensitive, non-invasive tool to quantitatively evaluate cerebrospinal fluid and interstitial fluid exchange in Type-2 diabetes mellitus and possibly in other neurological disorders, with potential clinical application.

Peripheral Levels of AGEs and Astrocyte Alterations in the Hippocampus of STZ-Diabetic Rats.

Diabetic patients and streptozotocin (STZ)-induced diabetes mellitus (DM) models exhibit signals of brain dysfunction, evidenced by neuronal damage and memory impairment. Astrocytes surrounding capillaries and synapses modulate many brain activities that are connected to neuronal function, such as nutrient flux and glutamatergic neurotransmission. As such, cognitive changes observed in diabetic patients and experimental models could be related to astroglial alterations. Herein, we investigate specific astrocyte changes in the rat hippocampus in a model of DM induced by STZ, particularly looking at glial fibrillary acidic protein (GFAP), S100B protein and glutamate uptake, as well as the content of advanced glycated end products (AGEs) in serum and cerebrospinal fluid (CSF), as a consequence of elevated hyperglycemia and the content of receptor for AGEs in the hippocampus. We found clear peripheral alterations, including hyperglycemia, low levels of proinsulin C-peptide, elevated levels of AGEs in serum and CSF, as well as an increase in RAGE in hippocampal tissue. We found specific astroglial abnormalities in this brain region, such as reduced S100B content, reduced glutamate uptake and increased S100B secretion, which were not accompanied by changes in GFAP. We also observed an increase in the glucose transporter, GLUT-1. All these changes may result from RAGE-induced inflammation; these astroglial alterations together with the reduced content of GluN1, a subunit of the NMDA receptor, in the hippocampus may be associated with the impairment of glutamatergic communication in diabetic rats. These findings contribute to understanding the cognitive deficits in diabetic patients and experimental models.

Alteration in amyloid ß42, phosphorylated tau protein, interleukin 6, and acetylcholine during diabetes-accelerated memory dysfunction in diabetic rats: correlation of amyloid ß42 with changes in glucose metabolism.

BACKGROUND: Diabetes accelerates memory dysfunction in a continuous, slowly pathological process. Studies suggest that the time course of certain biomarkers can characterize the pathological course of the disease to provide information for early intervention. Thus, there is an urgent need for validated biomarkers to characterize the cognitive impairment induced by DM. We aimed to detect changes in cerebrospinal fluid biomarkers such as amyloid ß42, phosphorylated tau protein, interleukin 6, and acetylcholine in diabetic rats over time, and to analyse the relationship between diabetes and cognitive impairment. METHODS: Rats were injected once intraperitoneally with 1% of streptozotocin to establish a diabetic model. Index changes were investigated longitudinally and all were measured at the end of the experiment at day 75. Aß42, P-tau, IL-6, and ACh levels in CSF, insulin levels in plasma, and Aß42 levels in plasma and brain tissue were measured by ELISA. RESULTS: Compared with control, the diabetic model showed ACh in CSF to be decreased by day 15, continuing lower out to day 75. Aß42 changes in brain and blood showed the same trends but exhibited minima at different time points: day 30 in CSF and day 15 in plasma. After the minimum, Aß42 in cerebrospinal fluid rose and levelled off lower than in the control group, whereas Aß42 in plasma rose and went above the controls at day 30, slowly trending upwards for the remainder of the experiment. P-tau protein in CSF in diabetic rats showed an increasing trend, becoming significantly different from the controls at day 60 and day 75. Aß42 in CSF was strongly negatively correlated with blood glucose at day 15 and was negatively correlated with insulin in serum, particularly at day 45. CONCLUSION: Our longitudinal research model suggest that changes in the measured biomarkers appear before learning and memory impairments do. Aß42 and ACh in the diabetes model group clearly changed from day 0 to day 45, and then P-tau and IL-6 varied significantly from day 45 to day 75. The reduced ACh levels observed possibly correlated with the factors common to changes in Aß42, P-tau, and IL-6.

Endoplasmic reticulum stress in the peripheral nervous system is a significant driver of neuropathic pain.

Despite intensive effort and resulting gains in understanding the mechanisms underlying neuropathic pain, limited success in therapeutic approaches have been attained. A recently identified, nonchannel, nonneurotransmitter therapeutic target for pain is the enzyme soluble epoxide hydrolase (sEH). The sEH degrades natural analgesic lipid mediators, epoxy fatty acids (EpFAs), therefore its inhibition stabilizes these bioactive mediators. Here we demonstrate the effects of EpFAs on diabetes induced neuropathic pain and define a previously unknown mechanism of pain, regulated by endoplasmic reticulum (ER) stress. The activation of ER stress is first quantified in the peripheral nervous system of type I diabetic rats. We demonstrate that both pain and markers of ER stress are reversed by a chemical chaperone. Next, we identify the EpFAs as upstream modulators of ER stress pathways. Chemical inducers of ER stress invariably lead to pain behavior that is reversed by a chemical chaperone and an inhibitor of sEH. The rapid occurrence of pain behavior with inducers, equally rapid reversal by blockers and natural incidence of ER stress in diabetic peripheral nervous system (PNS) argue for a major role of the ER stress pathways in regulating the excitability of the nociceptive system. Understanding the role of ER stress in generation and maintenance of pain opens routes to exploit this system for therapeutic purposes.

Insulin and glucagon share the same mechanism of neuroprotection in diabetic rats: role of glutamate.

In patients with acute ischemic stroke, diabetes and hyperglycemia are associated with increased infarct size, more profound neurologic deficits and higher mortality. Notwithstanding extensive clinical and experimental data, treatment of stroke-associated hyperglycemia with insulin is controversial. In addition to hyperglycemia, diabetes and even early prediabetic insulin resistance are associated with increased levels of amino acids, including the neurotoxic glutamate, in the circulation. The pleiotropic metabolic effects of insulin include a reduction in the concentration of amino acids in the circulation. In this article, we show that in diabetic rats exposed to transient middle cerebral artery occlusion, a decrease of plasma glutamate by insulin or glucagon reduces CSF glutamate, improves brain histology, and preserves neurologic function. The neuroprotective effect of insulin and glucagon was similar, notwithstanding their opposite effects on blood glucose. The therapeutic window of both hormones overlapped with the short duration (~30 min) of elevated brain glutamate following brain trauma in rodents. Similar neuroprotective effects were found after administration of the glutamate scavenger oxaloacetate, which does not affect glucose metabolism. These data indicate that insulin and glucagon exert a neuroprotective effect within a very brief therapeutic window that correlates with their capacity to reduce glutamate, rather than by modifying glucose levels.

Differential effects of diabetes on rat choroid plexus ion transporter expression.

Though diabetes is a disease with vascular complications, little is known about its effects on the blood-brain barrier or the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB is situated at choroid plexuses located in the lateral, third, and fourth ventricles. Choroid plexuses are the primary site of cerebrospinal fluid (CSF) production and express numerous ion transporters. Previous studies have shown a perturbation of ion transport in the periphery and brain during diabetes. In this study, we investigated the effect of diabetes on ion transporters in the choroid plexuses of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Sprague-Dawley rats by intraperitoneal injection of STZ (60 mg/kg in citrate buffer, confirmed by glucose analysis: 601 +/- 22 mg/dl diabetic rats, 181 +/- 46 mg/dl age-matched controls); and at 28 days, rats were killed, choroid plexuses harvested, and protein extracted. Western blot analyses were carried out using antibodies for ion transporters, including Na(+)-K(+)-2Cl(-) cotransporter and the Na(+)-K(+)-ATPase alpha1-subunit. The efflux of the K(+) analog (86)Rb(+) from choroid plexus was also studied. Diabetic rats showed an increase in expression of the Na(+)-K(+)-2Cl(-) cotransporter and the Na(+)-K(+)-ATPase alpha1-subunit, as compared with age-matched controls, a decrease in Na(+)-H(+) exchanger expression, and no change in Na(+)-K(+)-ATPase beta1- or beta2-subunit. The net effect of these changes was a 66% increase in (86)Rb(+) efflux from diabetic choroid plexus compared with controls. These changes in expression may affect choroid plexus ion balance and thus significantly affect CSF production in diabetic rats.

Elevated substance-P-like immunoreactivity levels in spinal dialysates during the formalin test in normal and diabetic rats.

Pharmacologic studies implicate the involvement of substance P in spinal nociceptive processing during the formalin test. However, no direct measurement of the temporal changes in substance P levels within the spinal cord of conscious animals has been reported. Further, dissociation between substance P levels and formalin-evoked nocifensive behavior may exist in diabetic rats, as exaggerated hyperalgesic behavior coexists with reduced peripheral nerve substance P levels. The present study was performed to directly measure the appearance of substance-P-like immunoreactivity (SP-LI) in spinal CSF of conscious, unrestrained rats using microdialysis techniques following injection of formalin into the hindpaw. The effect of diabetes upon formalin-evoked SP-LI levels in spinal CSF dialysates was also determined. In control rats, SP-LI increased in spinal dialysates following formalin injection and levels were maximal 20-30 min after injection, rising to 325% of basal values (p<0.02). Diabetic rats exhibited reduced (p<0.05) SP-LI in their spinal roots, while basal levels in spinal CSF were not different from controls. Formalin-evoked nocifensive behavior was increased in diabetic rats but SP-LI levels in spinal CSF dialysates after paw formalin injection were significantly (p<0.05) attenuated, reaching a maximum of only 161% of basal levels. This was accompanied by attenuated swelling at the formalin injection site and increased thermal response latencies. While increased SP-LI in spinal CSF coincides with phase 2 behavior in the formalin test and may contribute to spinal nociceptive processing during this period, exaggerated spinal substance P release is unlikely to underlie the increased nocifensive behavior seen in diabetic rats.

Uptake of circulating insulin-like growth factor-I into the cerebrospinal fluid of normal and diabetic rats and normalization of IGF-II mRNA content in diabetic rat brain.

Brain injury has been prevented recently by systemic administration of human insulin-like growth factor-I (hIGF-I). It is widely believed that protein neurotrophic factors do not enter the brain from blood, and the mechanism by which circulating hIGF-I may be neuroprotective is uncertain. This investigation tested the hypothesis that hIGF-I is taken up into cerebrospinal fluid (CSF) from the circulation. (125)I-hIGF-I was injected subcutaneously into rats. The (125)I-IGF-I recovered from CSF and plasma were indistinguishable in size from authentic (125)I-hIGF-I on SDS-PAGE. An ELISA was used that detected immunoreactive hIGF-I, but not rat IGF-I, rat IGF-II, human IGF-II, or insulin. Osmotic minipumps were implanted for constant subcutaneous infusion of various hIGF-I doses. Uptake into CSF reached a plateau at plasma concentrations above approximately 150 ng/ml hIGF-I; the plateau was consistent with carrier-mediated uptake. The plasma, but not CSF, hIGF-I level was significantly reduced in streptozotocin diabetic vs. nondiabetic rats, and uptake of hIGF-I into CSF was nonlinear with respect to plasma hIGF-I concentrations. Nonlinear uptake excluded leakage or transmembrane diffusion of IGF-I from blood into CSF as a dominant route for entry, but the site and mechanism of uptake remain to be established. The IGF-II mRNA content per milligram brain (P < 0.02) as well as per poly(A)(+) RNA (P < 0.05) was significantly increased towards normal in diabetic rats treated by subcutaneous administration of hIGF-I vs. vehicle. This effect of circulating hIGF-I may have been due to regulation of IGF-II gene expression in the choroid plexus and leptomeninges, structures at least in part outside of the blood-central nervous system barrier. These data support the hypothesis that circulating IGF-I supports the brain indirectly through regulation of IGF-II gene expression as well as by uptake into the CSF.