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_{2}) exposure using fractal analysis. _{2}) for 22.5 hours/day, and then 7 days in conditions of normoxia. Slaughtering of the rats was performed on day 21 and the eye globes were harvested in order to perform histopathological examinations. The fractal analyses of the retinal digital images were performed using the fractal analysis software Image J, and the fractal dimensions were calculated using the standard box-counting method. _{2}).

In the last few decades important efforts have been directed to understand various aspects of the newborn rats retinal layers, for clinical diagnostics and treatment [

The rat’s retina is immature at birth, being comparable with that from human fetuses of 26 weeks, and allows tracking its development postnatally [

Newborn rat model represents a solution to study normal and abnormal development of primary visual system of human subjects [

Computerized image visualization and advances in analysis methods of the retinal layers using the fractal geometry is a part of the early detection and diagnosis of retinal diseases and can be useful for describing the pathological architecture of retina [

The fractal analysis is applied to the fractal objects that cannot be measured properly using regular Euclidean geometry [

Fractal dimensions of image objects can be measured using computational approaches. Fractal dimension quantifies the degree of complexity into a single value, being a measure of how the fractal object fills up space. The fractal dimension measures a qualitative feature of fractal geometric objects that is used to distinguish different types of fractals.

The fractal analysis depends on the experimental and methodological parameters involved as diversity of subjects, image acquisition, type of image, image processing, fractal analysis methods, including the algorithm and specific calculation used, and so forth [

The experimental study was conducted at the “Iuliu Haţieganu” University of Medicine and Pharmacy Cluj-Napoca, in the Research Center of the Department of Physiology. The protocol was approved by the Ethics Committee of “Iuliu Haţieganu” University of Medicine and Pharmacy Cluj-Napoca [

In this study we used white rats, Wistar breed, females, and newborn rat 12 hours old, with birth weight of 10 grams. The animals came from Biobase of “Iuliu Haţieganu” University of Medicine and Pharmacy Cluj-Napoca and they were kept in vivarium conditions appropriate at the Department of Physiology. These were placed in an Isolette incubator, in which temperature was controlled at 23-24 degrees, light-dark cycle of 12 hours (using artificial white light 200 lux).

Females received a standard normocaloric diet, and water ad libitum and the newborn rats were fed by lactating mother. Toilet incubator was made for periods of

The experimental study was conducted on 20 animals divided into 2 equal lots for 21 days. Control group (normoxia) was performed in 10 newborn rats put on 12 hours of birth, along with their mother in an incubator, type Isolette, where they provided normoxic conditions for 21 days (21% O_{2}) (Days 0–21).

Consignment of animals exposed to hyperoxia was performed continuously in 10 rats put on 12 hours of birth, along with their mother in an incubator under conditions identical to those of control group (normoxia) for 7 days (Days 0–7).

In the next 7 days (Days 8–14) the animals were exposed to hyperoxia, daily to an oxygen concentration of 80% O_{2} for 22.5 hours/day. After exposure to hyperoxia, the incubator was again assured normoxic conditions for 7 days, from Day 15 until Day 21.

To achieve hyperoxic condition we used a mobile oxygen concentrator adapted to the incubator, with a flow rate = 1.5 L/min. Oxygen concentration was monitored twice/day using an electronic gas analyzer.

Processing of tissue samples and retinal histopathology were performed at the University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Laboratory of the Department of Histology, Cluj-Napoca. The histopathological examinations of the enucleated eyes were performed on 21 day. Serial sections of 5

In mathematical calculation, box counting or box dimension is the most common method used to estimate the fractal dimension [

The lower and upper box-counting dimensions of a subset

If these are equal then the common value is referred to as the box-counting dimension of

(if this limit exists), where

Twenty digital images were analyzed from each group. After adjustments of every digital image, a binarization on a binary image corresponding to the analyzed structure was obtained. The binary template, which is also referred to as structural element is associated to the binary image using logical operations [

In our study the box-counting algorithm was performed using the Image J software (Wayne Rasband, National Institutes of Health, in Bethesda, Maryland, USA) [

The algorithm was applied with the following options: (a) grid positions

The fractal dimension was calculated as the slope of the regression line for the log-log plot of the scanning box size and the count from a box-counting scan. The “count” usually refers to the number of grid boxes that contained pixels in a box-counting scan. The slope of the linear region of the plot is

The statistical processing of the results obtained with Image J software was done using GraphPad InStat software program, version 3.20 (GraphPad, San Diego, CA, USA) [

In the control group, all histological sections made showed a normal aspect of the retinal vessels and retinal layers (Figure

Control group (Goldner’s Trichrome ob. 40 X):

Retinal sections made of the animals exposed to hyperoxia showed structural abnormalities in both retinal periphery and center (Figure

O_{2} exposed group (Goldner’s Trichrome ob. 40 X):

Cytoarchitectural anomalies highlighted in this study emphasized the destructive effect on immature, developing retina after exposure to hyperoxia.

A summary of the obtained results is presented in Table

Results of the fractal dimensions (mean ± standard deviation) and correlation coefficients (

Retinal layer | Type | Fractal dimensions ( |
Correlation coefficients ( |
---|---|---|---|

Cells ganglionar layer CGL | N | 1.3250 ± 0.0123 | 0.9990 |

P | 1.2230 ± 0.0127 | 0.9980 | |

Internal plexiform layer IPL | N | 1.7196 ± 0.0121 | 0.9981 |

P | 1.6686 ± 0.0125 | 0.9980 | |

Internal nuclear layer INL | N | 1.7268 ± 0.0124 | 0.9986 |

P | 1.6048 ± 0.0123 | 0.9990 | |

Outer nuclear layer ONL | N | 1.7655 ± 0.0122 | 0.9981 |

P | 1.7437 ± 0.0125 | 0.9981 | |

Photoreceptors layer PL | N | 1.6912 ± 0.0126 | 0.9984 |

P | 1.5381 ± 0.0127 | 0.9953 |

Note: (

For all analyzed cases, the coefficients of correlation (

Cells ganglionar layer CGL (see Figure

Internal plexiform layer IPL (see Figure

Internal nuclear layer INL (see Figure

Outer nuclear layer ONL (see Figure

Photoreceptors layer PL (see Figure

The

The

The

The

The

Some remarks can be obtained concerning the results from Table

the average of fractal dimensions (

the fractal analysis was in agreement with the histological observations.

To obtain a more robust diagnostic tool, we think that fractal dimension together with other morphometric parameters could be an improved, powerful aid in understanding and discriminating pathological retinal layers. Based on this reason, the fractal dimension should be included in a global index considering different parameters.

The fractal analysis may describe the complexity of biological structures that cannot be sufficiently described using the classical Euclidian geometric terms [

The fractal analysis is valuable addition to quantify histoarchitectural changes in the newborn rats retinal layers during the hyperoxia (80% O_{2}).

Emerging data show that variation in the fractal dimensions may serve as an indicator or predictive factor in normal versus pathological conditions, serving as an objective means to guide the researchers.

The fractal analysis may be a tool for examining the mechanistic origins of pathological forms and may someday have a significant impact on our understanding of challenges in treatment delivery and diagnosis of retinal diseases.

None of the authors has a financial or proprietary interest in any material or method mentioned. The authors alone are responsible for the content and writing of the paper.