Seyyed Amirhossein Fazeli1, Anneh Mohammad Gharravi1, Soraya Ghafari1, Mehrdad Jahanshahi2, Mohammad Jafar Golalipour1

1Department of Embryology and Histology, Gorgan University of Medical Sciences, Gorgan, Iran
2Department of Anatomy, Gorgan University of Medical Sciences, Gorgan, Iran

Seyyed Amirhossein   Fazeli☆, M.D., Department of Embryology and Histology, Gorgan University of Medical Sciences, Gorgan, Iran

Corresponding author: Mohammad Jafar Golalipour, Ph.D., Professor, Department of Embryology and Histology, Gorgan University of Medical Sciences, Gorgan, Iran
mjgolalipour@yahoo.com

Supported by: a Research Grant from the Department of Research, Gorgan University of Medical Sciences*

www.crter.cn
www.nrronline.org

doi:10.3969/j.issn.1673-5374.2010.12.004

Abstract
BACKGROUND: Urtica dioica extract has been shown to play a protective role in the neurodegeneration associated with diabetes mellitus.
OBJECTIVE: To verify the neuroprotective efficacy of nettle extract on pyramidal cell density in the CA3 hippocampal subfield following administration of Urtica dioica extract to young diabetic rats.
DESIGN, TIME AND SETTING: A randomized, controlled, neurobiological study was performed at the Department of Histology and Embryology at the Gorgan University of Medical Sciences in Iran from 2006 to 2007.
MATERIALS: Urtica dioica leaves were collected from a cultivated plant in the suburb of Gorgan (northern Iran) and taxonomically identified by the Department of Pharmacognosy, Mazandaran University of Medical Sciences.
METHODS: A total of 20 male, albino, Wistar rats, aged 6–7 postnatal weeks, were randomly assigned to four groups: normal control, diabetic model, preventive, and treatment, with five rats in each group. Diabetes was induced by intraperitoneal injection of streptozotocin (80 mg/kg) in the diabetic and treatment groups. Rats from the preventive group received a hydroalcoholic extract of Urtica dioica (100 mg/kg per day) during the first 5 days, and then streptozotocin (80 mg/kg) was administered on day 6. One week following the streptozotocin injection, rats in the treatment group were intraperitoneally administered hydroalcoholic extract of Urtica dioica (100 mg/kg per day) for 4 weeks.
MAIN OUTCOME MEASURES: Following administration of Urtica dioica extract, the dorsal hippocampal formation of the right cerebral hemispheres was stained with cresyl violet. Area densities of CA3 pyramidal cells were measured.
RESULTS: The diabetic, preventive, and treatment groups exhibited reduced cell densities compared with the control group (P < 0.05). Moreover, densities of CA3 pyramidal cells in the treatment group were significantly reduced compared with the diabetic model group (P < 0.05).
CONCLUSION: The Urtica dioica extract exhibited no significant neuroprotective benefits in diabetes-induced loss of pyramidal cells in the CA3 hippocampal subfields of young diabetic rats.
Key Words: Urtica dioica; hippocampus; pyramidal cells; diabetes; neuroprotective efficacy

INTRODUCTION
   
Diabetes is a common disease that is often accompanied by learning and memory deficits[1-3]. Studies have suggested that hippocampal neuropathology is responsible for the cognitive deficits associated with diabetes[4-7]. Various neuroprotective measures have been proposed to restore the impaired cognitive functions and structural abnormalities accompanying diabetes. For example, antioxidant agents, such as mexiletine, etomidate, resveratrol, and sesamol, are considered to act as neuroprotective agents[8-11]. In addition, nifedipine, fluoxetine, and candesartan have been shown to improve di-abetes-associated cognitive impairment[12-14]. Moreover, Ginseng radix, Folium mori, and Anemarrhena rhizome are plant materials that have been shown to exhibit neuroprotective properties in diabetic encephalopathy[15-19].
Nettle leaf extract has been shown to exhibit neuroprotective effects in diabetic rats; therapeutic use of hydroalcoholic extract of stinging nettle (Urtica dioica) can compensate for granule cell loss in the diabetic rat dentate gyrus[20], which can ameliorate cognitive impairment in diabetes. In addition, Urtica dioica extract prevents astrogliosis in the dentate gyrus of diabetic rats[21}.
Urtica dioica, or stinging nettle, is an annual and perennial herb, characterized by stinging hairs[22]. In addition to reducing blood sugar levels[23], Urtica dioica also functions as an antioxidant[24-26]. An increasing number of studies have shown a pattern of selective neuronal vulnerability in the hippocampus. Neurons in the hilus/CA3
 
and CA1 hippocampal subfields appear particularly sensitive, whereas dentate gyrus granule cells appear resistant. However, the molecular basis for this differential susceptibility remains largely unknown[27].
The present study observed the influence of Urtica dioica on pyramidal cell density in the CA3 hippocampal subfield following streptozotocin (STZ)-induced diabetes in young rats.
  
MATERIALS AND METHODS

Design
A randomized and controlled neurobiological study.
Time and setting
The present study was performed at the Department of Histology and Embryology in Gorgan University of Medical Sciences in Iran from 2006 to 2007.
Materials
Animals
Twenty male, albino, Wistar rats, aged 6–7 weeks and weighing (158.0 ± 42.1) g, were provided by the Iranian Pasteur Institute. Rats were provided with a pellet diet and tap water ad libitum for full acclimatization. During experimentation, the animals were maintained in an air-conditioned room at (22 ± 2) °C under a 12-hour light/dark cycle. To prevent confounding effects on hippocampal histology, physical elements, such as toys, or mazes, were not available in the rat cages.
Approval was granted by the Animal Care and Ethics Committee of the Gorgan University of Medical Sciences.
Plant materials
Urtica dioica leaves were collected from the cultivated plant in a suburb of Gorgan (northern Iran) and tax-onomically identified by the Department of Pharmacognosy, Mazandaran University of Medical Sciences.
Main drugs and equipment

Methods
Preparation of Urtica dioica extract
The aerial parts of Urtica dioica were dissected into small pieces, dried in a circulating air stove, and ground to a powder. The powdered material was then macerated using a hydroalcoholic (60°) solvent for 48 hours. The ethanol was removed by vacuum distillation, and the resulting residue was filtered and concentrated at 40 °C, resulting in a gel material. In addition to thin-layer chromatography and purity tests (foreign matter, total ash, acid-insoluble ash, and water-insoluble ash) for qualification analysis, monosaccharide-linked reagent assay (spectrophotometry) was performed to determine polysaccharide concentrations in Urtica dioica leaves for extract standardization. Phytochemical analysis results revealed a high percentage of tannins, steroids, and low levels of flavonids, carotenoids, and saponins in the leaves of Urtica dioica.
At the time of administration, the prepared extract powder was dissolved in saline.  
Animal grouping and interventions
A total of 20 albino, Wistar rats, aged 6–7 weeks, were assigned to four groups (n = 5) as follows:  
Normal control group: no injection on the first day; daily saline injections during weeks 2–5.
Diabetic model group: STZ injection on the first day; daily saline injections during weeks 2–5.
Preventive group: intraperitoneal injection of hydroalcoholic Urtica dioica extract, 100 mg/kg per day, during the first 5 days; STZ injection on day 6, daily saline injections until end of experiment.
Treatment group: STZ injection on day 1; intraperitoneal injection of hydroalcoholic Urtica dioica extract,      100 mg/kg per day, during weeks 2–5.
To establish a hyperglycemic model on day 1, diabetes was induced with a single intraperitoneal injection of STZ (80 mg/kg) to overnight fasted rats.
Blood glucose measurements and glucose tolerance test
Blood samples for glucose measurements were collected from the tail vein of the 16-hour fasted rats. Blood glucose concentration was measured at the beginning, and weeks 1, 2, 4, and 5 using an ACCU-CHEK? Active Glucometer.
At the beginning of experimentation and after collecting blood samples, intraperitoneal glucose tolerance test was performed on the 16-hour fasted rats using 2 g dextrose/kg. Subsequently, STZ/Urtica dioica was administered. In all groups, blood samples were collected from the tail vein of fasted rats at 0, 30, 60, and 120 minutes after glucose load. Glucose tolerance test was also performed at week 5.
Tissue processing
At the end of week 5, 1 hour after measuring blood glucose and performing the glucose tolerance test, animals from the four groups were sacrificed following chloroform anesthesia. The rat skulls were dissected and brains were collected. The right hemispheres were then separated through the corpus callosum and were fixed in 10% formaldehyde for 48 hours, followed by paraffin embedding. The 7-μm thick coronal sections were serially collected from bregma –3.30 mm to –6.04 mm of the hippocampal formation[28] using a Leitz rotary microtome, with an interval of 20 μm between every two consecutive sections. The sections were stained with cresyl violet[29].
Morphometric study
Images of 20 selected coronal sections (serial sec-tions of anterior to posterior hippocampus) were collected using an Olympus BX 51 microscope and DP 12 digital camera, under 400 × magnification. An area of 62 500 μm2 was selected in the pyramidal layer of the CA3 hippocampus subfield in all sections. To measure CA3 pyramidal cell area density, images were transferred to a computer. Using OLYSIA Autobioreport software (Olympus Optical, Co. LTD, Tokyo, Japan), the appropriate grids were su-perimposed on the images and the cells were manually quantified. To perform unbiased measurements, the individual was double-blinded and only cells with significant characteristics of pyramidal cells were quantified.
Main outcome measures
CA3 pyramidal cell area densities.
Statistical analysis
Data was analyzed using SPSS statistical software version 11.5 (SPSS, Chicago, IL, USA). Results were expressed as Mean ± SD. Comparisons of measurement data were performed using one-way analysis of variance followed by LSD post hoc test (significant at P < 0.05).

RESULTS

Glucose tolerance test
At the beginning of the experiment, all groups exhib-ited normal glucose tolerance test result. At week 5, diabetic rats, which did not receive nettle extract, exhibited significantly impaired result of the test. The test result of the preventive group was less affected than rats of treatment group (Figure 1).   

Blood glucose levels
As shown in Figure 2, no significant hypoglycemic effects due to Urtica dioica extract were observed. In addition, blood glucose levels of the treatment group were greater than the diabetic and preventive groups at 1–4 weeks.
Pyramidal cell density and cresyl violet staining
One-way analysis of variance revealed that pyramidal cell density in the CA3 hippocampal region was significantly lower in groups with diabetes (diabetic, preventive, and treatment groups) than in the normal control group (P < 0.05) (Table 1).

LSD post hoc test demonstrated that cell density in the treatment group was significantly less than the normal control and diabetic model groups (P < 0.05).
Cresyl violet staining revealed that the number of CA3 hippocampal pyramidal cells corresponded to calculations of cell density in the same visual field      (1 000 ×) (Figure 3).

DISCUSSION

A recent study from our laboratory demonstrated granule cell loss within the dentate gyrus in young diabetic rats[20]. In addition, preventive administration of nettle extract resulted in no significant benefits. However, the therapeutic administration of Urtica dioica extract suggested some neuroprotective effects[20]. Results from the present study demonstrated pyramidal cell loss in the CA3 hip-pocampal subfield following STZ injection in rats be-tween 6–11 weeks of age. Surprisingly, therapeutic administration of nettle extract resulted in greater CA3 pyramidal cell loss.
Neuroprotective properties of the extract in the dentate gyrus have been attributed to antioxidant and an-ti-apoptotic properties of Urtica dioica[24-26]. These properties may be restricted to the dentate gyrus, which is a region of adult neurogenesis[30-31].
In summary, administration of Urtica dioica extract resulted in no neuroprotective benefits to CA3 py-ramidal cells in young diabetic rats.
 

 
Acknowledgments
We acknowledge the invaluable contributions made by Dr. Mohammad Hadi Soleimani and Mr. Naser Behnampour.

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 (Edited by Huang XJ, Wu ZZ/Yang Y/Song LP)