Gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) tracer-based magnetic resonance imaging (MRI) was employed to realize dynamic imaging and quantitative analysis of the diffusion of substances in the young and adult rat brain in vivo.

1. Experimental Animals:

The study was conducted in accordance with the established Chinese guidelines for the use of experimental animals, and the protocols were approved by the Ethics Committee of Peking University Health Science Center (Approval No. LA2012-016). 16 male Sprague Dawley rats were involved in the experiment: adult group ( n = 8) and postnatal 10 days old (P10) group (n = 8).

2. MRI Pre-Scanning:

Anesthetized rats (50 mg/kg sodium pentobarbital, intraperitoneal) were placed in a wrist coil for MRI pre-scanning to acquire a reference image. All rats were scanned in a 3.0-Tesla MRI system (Magnetom Trio; Siemens Medical Solutions, Erlangen, Germany) using a T1-weighted three-dimensional magnetization-prepared rapid acquisition gradient echo (T1 3D MP-RAGE) sequence. Parameters were as follows: echo time 3.7ms, repetition time 1500ms, flip angle 9°, inversion time 900ms, slice thickness 1mm, field of view 267mm, voxel 0.5×0.5× 0.5mm.¹

3. Intraparenchymal Microinjection:

Gd-DTPA (Magnevist; Bayer Schering Pharma AG, Berlin, Germany) was diluted with double distilled water into 10 mmol/L. The anesthetized rats were placed in a stereotaxic frame. The skull was opened using a surgical blade. For the whole brain volume of P10 is approximately equal to 60% of the adult, 2 µl and 1.2 µl of Gd-DTPA were respectively injected into the right striatum of adult and P10 rats with a 10µl Hamilton microsyringeµl at a speed of 0.2µl /min for 10 min, followed by a 5 min pause to avoid reflux along the needle track. The stereo coordinates are respectively 1 and 0.3 mm anterior from bregma, 3.5 and 2.3 mm lateral from median, 5.0 and 4.0mm deep.

4. MRI and Post-Processing:

MRI scanning was performed at predetermined time intervals after the intracranial injection of Gd-DTPA. The Statistical Parametric Mapping plug-in for MATLAB was used to coregister the MR images from the same rat pre-and post-injection.²

5. Calculation of Parameters:

In our previous study, a direct linear relationship between signal enhancement and Gd-DTPA concentration was observed under the same condition¹. Then the diffusion parameters within the extracellular space were calculated³: free diffusion coefficient (D), effective diffusion coefficient (D*), tortuosity (λ), and clearance rate constant (k’). The ratio of maximal volume of distribution (Vd_max) was also obtained.⁴

6. Immunohistochemical Analysis:

The immunofluorescence analysis was performed as previously described.⁵ Primary antibodies used were rabbit anti-Tenascin C (TN-C;1:250 dilution; Abcam, Cambridge, UK). The nuclei were counterstained with hematoxylin and eosin (HE).

7. Statistical Analysis:

Statistical analysis was performed by SPSS 19.0. Independent samplet-tests were used to test the difference of the parameters between the young and mature rats.

1. Comparison of the Gd-DTPA Diffusion Parameters

The signal intensity of the adult (Fig. 1A) and P10 (Fig.1B) rats striatum increased after the Gd-DTPA tracer was injected into the ECS. The hyperintensity then attenuated over time with the tracer distributing and clearence. Detailed datas were shown in Table 1. There were significant differences in the D* ( t =8.923;p=0.00) and tortuosity (λ=(D/D*)^1/2) between the two groups. Besides, a significant difference was observed in the k' (t =-4.919;p=0.01). However, the Vd_max showed no significant difference. Changes in the Gd-DTPA distribution volume in brain over time were shown in Figure 2.

2. Histological Characteristics and TN-C Expression

HE histological sections of the adult (Fig.3A) and P10 (Fig.3B) rat brain revealed patch compartments in the striatum. Immunohistochemistry for TN-C in the adult (Fig.3C) and P10 (Fig.3D) rat brain revealed more extensive expression of TN-C in P10 rat striatum.

The tracer-based MRI technique can not only achieve the multi-point measurements of diffusion parameters near the injection site, but also provide a 3-D visualization of the dynamic drainage flow of brain interstitial fluid.¹ Differences in the adult and P10 rat striatum ECS characteristics were observed in the D*, k’ and λ. TN-C is an extracellular matrix protein that is highly expressed in astroglial precursors in the early stage of life, which is associated with cell attachment and migration.^6,7 A former study has shown that TN-C-deficient mice have a significantly lower tortuosity.⁸ Therefore, we speculate that TN-C may play a role in the changing of ECS diffusion parameters during development. But further study is still needed.With the development of the rat brain, D*, k’ and λ of the striatum ECS has significant differences while the Vd_max shows no significant difference, which may be related to the expression quantity of TN-C.